Methods for the transformation of vegetal plastids

ABSTRACT

The present invention relates to novel methods for the generation of transgenic plants with genetically modified plastids, and to the transgenic plants generated with these methods.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP02/14302 filed Dec. 16, 2002, which claims the benefit ofGerman Application 101 63 161.8 filed Dec. 20, 2001.

FIELD OF THE INVENTION

The contents of the following submission on compact discs areincorporated herein by reference in its entirety: two copies of theSequence Listing (COPY 1 and COPY 2 REPLACEMENT Mar. 21, 2007) and acomputer-readable form of the Sequence Listing (CRF COPY REPLACEMENTMar. 21, 2007), all on CD-Rs, each containing: file name: SecondReplacement Sequence list-13173-00013-US, date recorded: Mar. 21, 2007,size: 95 KB.

The present invention relates to novel methods for the generation oftransgenic plants with genetically modified plastids and to thetransgenic plants generated using these methods.

DESCRIPTION OF THE BACKGROUND

Biotechnological work carried out on plants aims at generating plantswith advantageous novel properties, for example to increase agriculturalproductivity, to increase the quality in foodstuffs or for producingcertain chemicals or pharmaceuticals.

Plastids are organelles within plant cells which have their own genome.They play an essential role in photosynthesis and in amino acid andlipid biosynthesis. The plastids' genome consists of a double-stranded,circular DNA with an average size of from 120 to 160 kb and ispresent—for example in leaf cells—as approximately 1900 to 50,000 copiesper cell (Palmer (1985) Ann Rev Genet 19:325-54). A single plastid has acopy number of approximately from 50 to 100. The term plastids compriseschloroplasts, proplastids, etioplasts, chromoplasts, amyloplasts,leukoplasts and elaioplasts (Heifetz P (2000) Biochimie 82:655-666). Thevarious forms can be converted into one another and all arise from theproplastids. This is why all manifested forms of the plastids comprisethe same genetic information. Preference is given in the literature asstarting material for the transformation of plastids, however, to greencells, which comprise the chloroplasts as the manifested form.

It is of great economic interest for plant biotechnologists to developefficient methods for the transformation of plastids (McFadden G (2001)Plant Physiol. 125:50-53). The stable transformation of plastids ofhigher plants is one of the great challenges.

In the transformation of plastids, the technique of undirected(illegitimate) DNA insertion, which is frequently employed in insertioninto the nuclear DNA, has the disadvantage that it is highly likely thatan essential gene on the gene-dense plastidic genome is affected, whichwould frequently be lethal for the plant. The directed insertion offoreign DNA is therefore advantageous in plastids.

Various methods for the directed insertion into the plastidic genomehave been described. The first to be described was plastidtransformation in green algae (Boynton J E et al. (1988) Science 240:1534-1538; Blowers A D et al. (1989) Plant Cell 1:123-132), followedlater by higher plants such as tobacco (Svab Z et al. (1990) Proc NatlAcad Sci USA 87:8526-8530).

EP-A 0 251 654, U.S. Pat. Nos. 5,932,479, 5,451,513, 5,877,402, WO01/64024, WO 00/20611, WO 01/42441, WO 99/10513, WO 97/32977, WO00/28014, WO 00/39313 describe methods and DNA constructs for thetransformation of plastids of higher plants, where the DNA to betransformed is introduced into the plastome (plastidic genome) viahomologous recombination (“double crossover”). In general, homologousregions of 1000 bp or more on either side of the sequence to be insertedare employed. This rapidly gives rise to large vectors whose handling isnot very convenient. Moreover, the transformation efficiency drops. Thehomologous recombination efficiency drops with the increasing length ofthe foreign DNA to be integrated. A further disadvantage is the factthat a homologous region which can be utilized for the process of DNAintegration by means of double crossover must be identified for eachplant species. WO 99/10513 claims the identification of an intergenicDNA sequence with supposedly sufficient homology between the genomes ofthe chloroplasts of many higher plants, which DNA sequence can thus actas a universal target sequence. However, it has not been demonstratedthat this vector can be utilized successfully in species other thantobacco; rather, in WO 01/64024, the same inventor adapts thetransformation vector to non-tobacco plant species by using homologousDNA sequences isolated from these plant species. Since only fewrecombination events result in all of the above-described methods,selection of the recombinant plastidic DNA molecules is required.

The plastid DNA of higher plants is present in the form of up to severalthousand copies per cell. To ensure stable integration of foreign DNA,all copies of the plastidic DNA must be modified in the same manner. Inplastid transformation, this is referred to as having reached thehomotransplastomic state. This state is achieved by what is known as asegregation-and-sorting process, by exerting a continuous selectionpressure on the plants. Owing to the continual selection pressure, thoseplastids in which many copies of the plastidic DNA have already beenmodified are enriched during cell and plastid division. The selectionpressure is maintained until the homotransplastomic state is reached(Guda C et al. (2000) Plant Cell Reports 19:257-262). The modificationof all of the copies of the plastidic genome in order to obtainhomotransplastomic plants which have incorporated the foreign genestably into their plastidic genome over generations without addition ofa selection agent is a great challenge (Bogorad L (2000) TIBTECH18:257-263). In addition to the continuous selection pressure, achievingthe homotransplastomic state is, if appropriate, ensured by repeatedlyregenerating tissue which has already been transformed (Svab Z andMaliga P (1993) Proc Natl Acad Sci USA 90:913-917). However, thisprocedure limits the plant material which is available for plastidtransformation. Coupling, if appropriate, the transgene with anothergene which is essential for the survival of the plant is thereforeproposed.

In most cases, tissue culture techniques and selection processes cannotbe applied universally to all plant species and constitute a substantiallimitation of plastid transformation, in particular with regard to theapplicability of the method to species other than tobacco. (Kota M etal. (1999) Proc Natl Acad Sci USA 96:1840-1845). A recently publishedtransformation of tomato plastids is based on modifications in theregeneration and selection scheme (Ruf S et al. (2001) Nature Biotech19:870-875), which, however, are expensive and time-consuming. Anotherapproach aims at reducing the number of plastids per cell and the DNAmolecules per plastid so that fewer DNA molecules have to be modified(Bogorad L (2000) TIBTECH 18:257-263). All of the selection andsegregation processes are very time-consuming.

WO 99/10513 describes a method in which a plastidic ORI (origin ofreplication) is localized on the plasmid to be transformed in order toincrease, in this manner, the number of copies of the vector to betransformed which are available for integration into the plastidicgenome (Guda C et al. (2000) Plant Cell Reports 19:257-262).

The necessity of improving the plastid transformation technique is alsomentioned in Heifetz and Tuttle (Heifetz P and Tuttle A M (2001) CurrOpin Plant Biol 4:157-161). WO 00/32799 teaches increasing theefficiency of plastid transformation by employing plants with enlargedplastids. This results in a large plastid surface, through which the DNAto be transformed can enter the plastids with greater ease. However, themechanism of DNA integration relies, again, on conventional homologousrecombination, as was the case in the above-described methods.

A variety of other methods for the sequence-specific integration ofDNA—in particular into the nuclear DNA—have been described. A methodbased on self-splicing group II introns has been described.Self-splicing group II introns are capable of inserting in asequence-specific fashion, for example into intron-free genes. Thesequence-specific hydrolysis of the target DNA is catalyzed by anRNA-protein (ribonucleoprotein) complex. Here, the sequence specificityof the endonuclease function is determined in particular by basepairings being formed between the RNA moiety of the ribonucleoproteincomplex and the target DNA. The use of group II introns as vectors forforeign DNA has been discussed. By modifying certain sequences of agroup II intron, it was possible to modify the target specificity of thelatter. Also, it was possible to insert further sequences into group IIintrons without destroying functions of the latter (Yang J et al. (1996)Nature 381:332-335; Eickbush T H (1999) Curr Biol 9:R11-R14; Matsuura Met al. (1997) Genes Develop 11:2910-2924; Cousineau B et al. (1998) Cell94: 451-462). The adaptation to certain target sequences and thedetermination of the associated rules, however, is laborious and has asyet been elucidated in detail only for the Ll.ltrB intron (Mohr G et al.(2000) Genes Develop 14:559-573). Moreover, the retrohoming efficiencywas reduced significantly by the modification, and not every single oneof the modified introns tested inserted into the desired target DNA. Thedisadvantage of the technique is that some positions in the nucleotidesequence are fixed, which limits the choice of the target region in theDNA to be transformed (Guo H et al. (1997) EMBO J. 16:6835-6848).Moreover, the efficiency of the retrohoming process with regard to thatof the wild-type intron appears to be diminished. The efficiency ofintron insertion at different sites on the genes investigated differedwith regard to its level. The work aimed at providing an improved methodfor the directed insertion of DNA into the nuclear DNA of organismswhich permit no efficient homologous recombination (Guo et al. (2000)Science 289:452-456). The experiments described have been carried outextrachromosomally both in the prokaryote E. coli and in human cells.The applicability to the chromosomal DNA of higher organisms or theapplicability to plastidic DNA was neither described nor demonstrated.It was merely proposed to attempt the optimization of this system insuch a way that insertion into chromosomal DNA of higher eukaryotes cantake place. This system is supposed to be an alternative method forhigher eukaryotes which lack efficient homologous recombination (Guo etal. (2000) Science 289:452-456). This does not apply to plastids ofhigher plants, where homologous recombination—at least in the case ofindividual plastidic DNA molecules—can usually be performed withoutproblems.

Plastid transformation was demonstrated not only in tobacco, but also inpotato (Sidorov V A et al. (1999) Plant J 19:209-216; WO 00/28014),petunia (WO 00/28014), rice (Khan M S and Maliga P (1999) Nature Biotech17:910-915; WO 00/07431; U.S. Pat. No. 6,153,813), Arabidopsis (Sikdar SR et al. (1998) Plant Cell Reports 18: 20-24; WO 97/32977) and oilseedrape (WO 00/39313). (Review article: Bogorad L (2000) TIBTECH18:257-263). Transplastomic tomato plants have also been describedrecently (Ruf S et al. (2001) Nature Biotech 19:870-875).

The generation of sequence-specific double-strand breaks with the aid ofrestriction enzymes in eukaryotic genomes, including plants, has beendescribed (Puchta H (1999) Methods Mol Biol 113:447-451).

WO 96/14408 describes the homing restriction endonuclease I-SceI andvarious possibilities for its use. An application for inserting DNAsequences into plastidic DNA is not described.

Posfai et al. describe a method for the substitution of genes in theprokaryote E. coli (Posfai G et al. (1999) Nucleic Acids Res27(22):4409-4415). Here, an intramolecular recombination between theendogenous and the mutated gene takes place in the E. coli genome, whichcombination is induced by cleaving with the restriction enzyme I-SceI.Recombinations in E. coli proceed markedly more efficiently and,presumably, following different mechanisms than is the case in thenucleus of higher eukaryotes (for example described by Kuzminov A (1999)Microbiol Mol Biol Rev. 63(4):751-813).

“Homing” refers to the phenomenon that two or more copies of a DNAsequence exist in one compartment, where at least one of these twosequences is interrupted by a further DNA sequence, and a copy of theinterrupting DNA sequence is subsequently also introduced into thenoninterrupted DNA sequence. This phenomenon usually takes the form ofintron homing. Here, two or more alleles of one gene exist in onecompartment, where at least one of these alleles has no intron. A copyof the intron is subsequently also introduced into the intron-freeallele.

Introns in plastidic genes of higher plants have been described (Vogel Jet al. (1999) Nucl Acids Res 27:3866-3874; Jenkins B D et al. (1997)Plant Cell 9:283-296; Xu M Q et al. (1990) Science 250: 1566-1570). Thesplicing of a homologous, unmodified intron with the natural exonregions at an ectopic locus in the plastidic genome has likewise beendescribed (Bock R and Maliga P (1995) Nucl Acids Res 23(13):2544-2547).Experiments of introducing, into plastids of higher plants, heterologousintrons which are additionally modified in such a way that they compriseadditional genetic information and/or splice in a normatural sequenceenvironment have not been carried out as yet.

Experiments carried out by Eddy and Gold into the homing process in E.coli have demonstrated that certain recombination systems are required.The type of the recombination system of the host is a key variable (EddyS R and Gold L (1992) Proc Natl Acad Sci USA 89:1544-1547). It wastherefore impossible to assume that the naturally occurring homingprocess of one organism can be applied at will to another organism, inparticular when this process probably does not occur naturally in thelatter organism.

Dürrenberger et al. describe the induction of an intrachromosomalrecombination in chloroplasts of the single-celled green algaChlamydomonas reinhardtii using the I-CreI homing endonuclease(Dürrenberger F et al. (1996) Nucleic Acid Res 24(17):3323-3331).

The recombination takes place between the endogenous 23S gene and a23S-cDNA which is inserted into the chromosome of an I-CreI deletionstrain and which comprises an I-CreI cleavage site. Double-strand breaksare induced by mating the relevant transgenic organism with an organismwhich naturally expresses I-CreI. At the point in time of thedouble-strand break, the foreign DNA is already inserted into thechromosomal DNA, and recombination takes place intramolecularly and notbetween two separate molecules.

It has been shown recently that a mobile intron which naturally occursin Chlamydomonas reinhardtii and which also encodes a homingendonuclease can be transformed efficiently into an intron-free copy(Odom O W et al. (2001) Mol Cell Biol 21: 3472-3481). In this work, theincrease of the transformation rate was dependent on the presence of thehoming endonuclease. In the discussion, it is proposed in general termsand without specific suggestions regarding the implementation, toimprove plastid transformation by inducing double-strand breaks. To thisend, the recognition regions of rare nucleases were initially to beintroduced in a first step, and the subsequent integration event wasthen to take place at the same locus. More detailed suggestionsregarding the manner in which the recognition regions are to beintroduced, the type of nucleases and recognition regions which can beused, the way in which the first step and the second step can bedesigned in actual reality, and the like, are not provided. All that hasbeen shown to date is that the introduction of a homologous intron, intoplastids of the alga Chlamydomonas, by means of the homing endonucleasenaturally associated with the mobility of the intron did work. Moreover,the results were generated in an algal species. The abovementionedexperiments by Eddy and Gold with E. coli, where no mobile group Iintrons are known, as is the case with the plastids of higher plants,demonstrate that an applicability to heterologous systems is not readilyfeasible. It is therefore by no means obvious for the skilled worker toapply the observations on the alga Chlamydomonas to higher plants. Incontrast, there are a number of suggestions which make such anapplicability rather doubtful:

-   1. Homing systems cannot be applied readily from one system to    another (Eddy S R and Gold L (1992) Proc Natl Acad Sci USA    89:1544-1547). The applicability to higher plants is all the more    dubious since no homing endonucleases have been identified in those    plastidic genomes of higher plants which have already been sequenced    (http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html).    It can therefore be assumed that the introns found in the plastidic    genome of higher plants are not mobile, and that no homing mechanism    exists naturally in these genomes.-   2. Chlamydomonas only has one plastid per cell, while in cells of    higher plants up to 100 plastids are present per cell.-   3. The efficiency of conventional plastid transformation in    Chlamydomonas exceeds that in higher plants by several orders of    magnitude, which suggests that these two systems cannot be compared    directly with one another. As regards the regeneration of    transplastomic algae or transplastomic plants, the fact that    division of the algal plastids is synchronized with the cell cycle,    while this is not the case for the plastids of the higher plants,    might also play an important role (Sato N (2001) Trends Plant    Science 6:151-155).-   4. The mechanisms of DNA integration into plastids of Chlamydomonas    and of higher plants appear to be fundamentally different. Thus, it    has been found that inter-specific plastid transformation (where    homologous regions are utilized instead of identical sequences) in    Chlamydomonas leads to a marked reduction of the transformation    efficiency, which was, however, not observed in tobacco. This also    applies analogously to the distance of a molecular marker on the    homologous DNA from the heterologous sequence on the transformation    plasmid: the closer the molecular marker to the edge of the target    region for integration by means of double crossover, the less    frequently it is transferred when transformed into Chlamydomonas    plastids. In tobacco, multiple recombination mechanisms were    observed, but here even molecular markers which were close to the    edge of the homologous regions were transferred efficiently into the    plastidic genome during transformation (Kavanagh TA et al. (1999)    Genetics 152: 1111-1122 and references cited therein).-   5. In Chlamydomonas, the plastids of the two parents fuse during    hybridization, even in the case of inter-specific hybridization. In    Chlamydomonas, plastid fusion is a natural process, and the DNA of    the plastids too is mixed and undergoes new recombination. This is    why mobile introns in the organelles of these organisms make sense.    In contrast, in most of the higher plants, the plastids are    inherited uniparentally, so that neither mixing of the plastidic DNA    results nor recombinations can occur between the maternal and the    paternal plastidic DNA. Even in those plant species in which the    plastids are inherited biparentally, no plastid fusion was observed.    It can therefore be assumed that natural plastid fusion in higher    plants can be ruled out (Hagemann R (1992) plastidic genetics in    higher plants; in Cell organelles, editor: Herrmann R G, Springer    Verlag, Vienna, pp. 65-96) and that mechanisms like intron homing    are either not developed or even suppressed.

Increasing the homologous recombination efficiency within the nuclearDNA with the aid of rare endonucleases has been described for variousorganisms (Puchta H et al. (1993) Nucleic Acids Research.21(22):5034-40; Puchta H et al. (1996) Proc Natl Acad Sci USA93:5055-5060; Rong Y S and Golic K G (2000) Science 28:2013-2018; JasinM (1996) Trends Genet 12: 224-229). In contrast to plastids, insertionby homologous recombination into the nuclear DNA is problematic andusually takes place owing to random illegitimate integration. Thisdemonstrates that techniques which are established for the nucleargenome cannot necessarily be applied to the plastids. In contrast to thesituation regarding the nucleus, integration in plastids of higherplants takes place virtually exclusively, and with high efficiency, viahomologous recombination (Bock R and Hagemann R (2000) Progress inBotany 61:76-90; Maliga P et al. (1994) Homologous recombination andintegration of foreign DNA in plastids of higher plants. In Homologousrecombination and gene silencing in plants. Paszkowski J, ed. (KluwerAcademic publishers), pp. 83-93).

The homologous recombination efficiency for the integration of DNA intothe plastome has generally not been thought of as a limiting factor and,in contrast, considered as not being critical. Accordingly, currentresearch into the optimization of plastid transformation does not focuson the optimization of homologous recombination but for example onimproved selection markers, improved selection and regenerationtechniques and the like. Nevertheless, no essential breakthrough hasbeen achieved to date.

SUMMARY OF THE INVENTION

As emphasized clearly by the above-described methods and problems in thetransformation of plastids, providing novel methods for the generationof homotransplastomic plants is a long-existing, unmet need of plantbiotechnology. A further need is the avoidance of antibiotic orherbicide selection markers for reasons of registration and consumeracceptance. To date, no plastid transformation method has been describedwhich does away with the need for such a selection marker.

It is therefore an object to develop novel methods which ensureefficient integration of foreign DNA in all copies of the plastidic DNAand which make possible the efficient selection of correspondinghomotransplastomic plants. Surprisingly, this object has been achievedby providing the integration/selection method according to theinvention.

A first subject matter of the invention relates to a method for theintegration of a DNA sequence into the plastidic DNA of a multi-celledplant or cell derived therefrom and for the selection of predominantlyhomotransplastomic cells or plants, wherein

-   a) the plastidic DNA molecules of said multi-celled plant or cell    derived therefrom comprise at least one recognition sequence for the    directed induction of DNA double-strand breaks and-   b) at least one enzyme suitable for the induction of DNA    double-strand breaks at the recognition sequence for the directed    induction of DNA double-strand breaks and at least one    transformation construct comprising an insertion sequence are    combined in at least one plastid of said multi-celled plant or cell    derived therefrom, and-   c) DNA double-strand breaks are induced at the recognition sequences    for the directed induction of DNA double-strand breaks, and-   d) the insertion sequence inserts into the plastidic DNA, the    functionality of the recognition sequence for the directed induction    of DNA double-strand breaks being deactivated so that said    recognition sequence is no longer capable of being cleaved by the    enzyme suitable for the induction of DNA double-strand breaks, and-   e) plants or cells in which the insertion sequence has been inserted    into the plastidic DNA molecules are isolated.

Surprisingly, the system makes possible a substantial increase of heefficiency in the generation of predominantly homotransplastomic plants.In this context, not only the efficacy of insertion into the plastidicDNA, but also the efficacy of the selection process of predominantlyhomotransplastomic plants are increased.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE INVENTION

Application of the method according to the invention results in aselection pressure of incorporating the insertion sequence into all ofthe copies of the plastidic DNA. Ideally, the insertion sequence isspread independently of the selection markers, such as herbicide orantibiotic resistances. This has pronounced advantages with regard toregistration and/or consumer acceptance. However, the use of suchselection markers can further increase the efficiency. The methodaccording to the invention for the generation of homoplastomic plantsclearly outperforms the prior-art methods since it shows a more rapid,more efficient and therefore more economical route to obtainhomotransplastomic plants. A further advantage of the system is that thesize of the constructs employed for the transformation can be kept smallsince the homologous regions in the plastid transformation vector can besmaller in comparison with the integration by means of double crossover,or can be completely absent.

The transformation of plastids has a large number of advantages over thetransformation of the nucleus. The following are to be mentioned interalia:

-   a) While homologous recombination into the nuclear DNA can only be    realized with difficulty, DNA in plastids can be integrated readily    at a predefined locus by means of double crossover, a form of    homologous recombination. Positional effects or gene silencing,    which are encountered in transformations of the nucleus owing to the    illegitimate integration at a non-predefined locus, are thus    avoided.-   b) Very high expression levels can be achieved, presumably owing to    the high copy number of the plastidic DNA.-   c) In higher plants, plastidic DNA is, as a rule, only subject to    maternal inheritance so that the foreign DNA introduced cannot be    spread via pollen and cross-pollination can thus be prevented    effectively.-   d) The prokaryotic nature of the plastids makes possible the    expression of genes in the context of a polycistronic operon    structure. It is therefore not necessary to equate each gene to be    expressed with its own promoter and the like. This facilitates the    introduction of a large number of genes in one pass, for example for    introducing entire biosynthetic pathways into the plastids.

“Plastid” refers to the proplastids and to all organelles to which theygive rise, such as, for example, chloroplasts, etioplasts, chromoplasts,amyloplasts, leukoplasts, dermaplasts and elaioplasts (Heifetz P (2000)Biochimie 82:655-666).

“Plastome” refers to the genome, i.e. the totality of the geneticinformation, of a plastid.

“Homotransplastomic” refers to a transplastomic and homoplastomic state.

With regard to, for example, a plant, cell, tissue, plastid or aplastidic DNA, “transplastomic” refers to all those forms of the above,realized by recombinant methods, which comprise a plastidic DNA whichhas been modified by recombinant methods, it being possible for themodification to comprise, for example, substitutions, additions,deletions, inversions or insertions of one or more nucleotide residues.

“Heteroplastomic” refers to the presence of a mixed population of avariety of plastidic DNAs within a single plastid or within a populationof plastids within a plant cell or tissue.

“Homoplastomic” refers to a uniform population of plastidic DNA within asingle plastid or within a population of plastids within a plant cell ortissue. Homoplastomic cells, tissues or plants are genetically stablesince they only comprise one type of plastidic DNA, i.e. they generallyremain homopolastomic even when the selection pressure ceases. Progenyobtained by selfing are likewise homoplastomic.

For the purposes of the present invention, “predominantly homoplastomic”or “predominantly homotransplastomic” refers to all those plants orcells in which the percentage of the desired plastidic DNA moleculeswhich have been modified with regard to a trait—for example with therecognition sequence for the directed induction of DNA double-strandbreaks or the inserted insertion sequence—amounts to at least 50%,preferably at least 70%, very especially preferably at least 90%, mostpreferably at least 95% of the totality of all plastidic DNA moleculesin a plant or a tissue, cell or plastid of same. Predominantlyhomoplastomic or predominantly homotransplastomic plants can beconverted into homoplastomic or homotransplastomic plants by continuedmaintenance of the selection pressure and, if appropriate, repeatedregeneration steps. Owing to the homing process, however, a continuousselection pressure is not necessarily required. In a particularembodiment, a predominantly homoplastomic, or homotransplastomic, plantis therefore truly homoplastomic, or homotransplastomic. A plant which,with regard to a DSB recognition sequence, is predominantlyhomoplastomic or homotransplastomic, or truly homoplastomic orhomotransplastomic, is subsequently referred to as “master plant”. Thepercentage of the desired plastidic DNA molecules which have beenmodified with regard to a trait can be determined in the manner known tothe skilled worker, for example by means of Southern analysis asdescribed by way of example in Example 4. The ratio between the plastidstarting DNA molecules and the plastidic DNA molecules which have beenmodified with regard to a trait can be determined by comparing theintensity of the bands in question.

“Multi-celled plant or cell derived therefrom” refers generally to allthose cells, tissues, parts or propagation materials (such as seeds orfruits) of a plant which constitutes, or may constitute, a multi-celledorganism in its adult state. Included for the purpose of the inventionare all genera and species of higher and lower plants of the plantkingdom. Annual, perennial, monocotyledonous and dicotyledonous plantsare preferred. Included are mature plants, seeds, shoots and seedlings,and parts derived therefrom, propagation material (for example tubers,seeds or fruits) and cultures, for example cell or callus cultures.“Mature plants” means plants at any developmental stage beyond theseedling stage. The term seedling means a young immature plant an earlydevelopmental stage.

Preferred plants are those from the following plant families:Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae,Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae,Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae,Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae,Tetragoniaceae, The aceae, Umbelliferae.

Preferred monocotyledonous plants are selected in particular from themonocotyledonous crop plants such as, for example, the Gramineae familysuch as rice, maize, wheat or other cereal species such as barley,millet and sorghum, rye, triticale or oats, and sugar cane, and allgrass species.

Preferred dicotyledonous plants are selected in particular fromdicotyledenous crop plants, such as, for example,

-   -   Asteraceae such as sunflower, tagetes or calendula and others,    -   Compositae, especially the genus Lactuca, very particularly the        species sativa (lettuce) and others,    -   Cruciferae, particularly the genus Brassica, very particularly        the species napus (oilseed rape), campestris (beet), oleracea cv        Tastie (cabbage), oleracea cv Snowball Y(cauliflower) and        oleracea cv Emperor (broccoli) and other cabbages; and the genus        Arabidopsis, very particularly the species thaliana, and cress        or canola and others,    -   Cucurbitaceae such as melon, pumpkin/squash or zucchini and        others,    -   Leguminosae, particularly the genus Glycine, very particularly        the species max (soybean), soybean, and alfalfa, pea, bean or        peanut and others,    -   Rubiaceae, preferably the subclass Lamiidae such as, for        example, Coffea arabica or Coffea liberica (coffee bush) and        others,    -   Solanaceae, particularly the genus Lycopersicon, very        particularly the species esculentum (tomato), the genus Solanum,        very particularly the species tuberosum (potato) and melongena        (aubergine) and tobacco or paprika and others,    -   Sterculiaceae, preferably the subclass Dilleniidae such as, for        example, Theobroma cacao (cacao bush) and others,    -   Theaceae, preferably the subclass Dilleniidae such as, for        example, Camellia sinensis or Thea sinensis (tea shrub) and        others,    -   Umbelliferae, particularly the genus Daucus (very particularly        the species carota (carrot)) and Apium (very particularly the        species graveolens dulce (celery)) and others; and the genus        Capsicum, very particularly the genus annuum (pepper) and        others,        and linseed, soybean, cotton, hemp, flax, cucumber, spinach,        carrot, sugar beet and the various tree, nut and grapevine        species, in particular banana and kiwi fruit.

Also encompassed are ornamental plants, useful or ornamental trees,flowers, cut flowers, shrubs or turf. Plants which may be mentioned byway of example but not by limitation are angiosperms, bryophytes suchas, for example, Hepaticae (liverworts) and Musci (mosses);pteridophytes such as ferns, horsetails and clubmosses; gymnosperms suchas conifers, cycads, ginkgo and Gnetatae, the families of the Rosaceaesuch as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceaesuch as poinsettias and croton, Caryophyllaceae such as pinks,Solanaceae such as petunias, Gesneriaceae such as African violet,Balsaminaceae such as touch-me-not, Orchidaceae such as orchids,Iridaceae such as gladioli, iris, freesia and crocus, Compositae such asmarigold, Geraniaceae such as geranium, Liliaceae such as dracena,Moraceae such as ficus, Araceae such as philodendron and many others.

Most preferred are Arabidopsis thaliana, Nicotiana tabacum, Tagetes andBrassica napus and all those genera and species which are used as foodsor feeds, such as the above-described cereal species, or which aresuitable for the production of oils, such as oil plants, nut species,soybean, sunflower, pumpkin/squash and peanut.

“Enzyme suitable for inducing DNA double-strand breaks at therecognition sequence for the directed induction of DNA double-strandbreaks” (hereinbelow referred to as “DSBI enzyme” for “doublestrand-break inducing enzyme”) generally refers to all those enzymeswhich are capable of generating, in a sequence-specific manner,double-strand breaks in double-stranded DNA. The following may bementioned by way of example, but not by limitation:

-   1. Restriction endonucleases, preferably type II restriction    endonucleases, especially preferably homing endonucleases as    described in detail hereinbelow.-   2. Artificial nucleases such as described in detail hereinbelow,    such as, for example, chimeric nucleases, mutated restriction or    homing endonucleases, or RNA protein particles derived from mobile    group II introns.

Both natural and artificially generated DSBI enzymes are suitable.Preferred are all those DSBI enzymes whose recognition sequence is knownand which can be obtained either in the form of their proteins (forexample by purification) or which can be expressed using their nucleicacid sequence.

The DSBI enzyme, whose specific recognition sequence is known, ispreferably selected in such a way that it has no further functionalrecognition regions in the plastidic genome, in addition to the targetrecognition sequence. Homing endonucleases are therefore very especiallypreferred (review: Belfort M and Roberts R J (1997) Nucleic Acids Res25:3379-3388; Jasin M (1996) Trends Genet 12:224-228; website:http://rebase.neb.com/rebase/rebase.homing.html; Roberts R J and MacelisD (2001) Nucleic Acids Res 29: 268-269). These meet this requirementowing to their long recognition sequences. Owing to the small size ofthe plastome, however, it is also feasible that DSBI enzymes withshorter recognition sequences (for example restriction endonucleases)can be employed successfully.

In addition to the above-described preferred embodiment, where only asingular recognition sequence for the DSBI enzyme is present in theplastidic DNA, cases where further, functionally identical, recognitionsequences can be employed advantageously are also feasible. This is thecase in particular when the plastome comprises duplicated genes (forexample in the form of inverted repeats). Here, integration into allcopies is to take place, so that cleavage in all copies is likewisedesirable.

The sequences which encode such homing endonucleases can be isolated forexample from the chloroplast genome of Chlamydomonas (Turmel M et al.(1993) J Mol Biol 232: 446-467). They are small (18 to 26 kD), but have,in their open reading frame (ORF), a “coding usage” which is directlysuitable for expression in the nucleus or plastids of higher plants(Monnat R J Jr et al. (1999) Biochem Biophys Res Com 255:88-93).

Further homing endonucleases are mentioned in the abovementionedwebsite; homing endonucleases which may be mentioned are, for example,F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-CeuI,I-CeuAIIP, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CreI, I-CrepsbIP,I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-CsmI, I-CvuI, I-CvuAIP,I-DdiII, I-DirI, I-DmoI, I-HspNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI,I-NclIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP,I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PpbIP, I-PpoI,I-SPBetaIP, I-ScaI, I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI,I-SceVII, I-SexIP, I-SneIP, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP,I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiS3bP, I-TdeIP, I-TevI,I-TevII, I-TevIII, I-UarAP, I-UarHGPAlP, I-UarHGPA13P, I-VinIP, I-ZbiIP,PI-MtuI, PI-MtuHIP, PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII,PI-PspI, PI-Rma438121P, PI-SPBetaIP, PI-SceI, PI-TfuI, PI-TfuII,PI-Thyl, PI-TliI, PI-TliII.

Preferred in this context are those homing endonucleases whose genesequences are already known, such as, for example, F-SceI, I-CeuI,I-ChuI, I-DmoI, I-CpaI, I-CpaII, I-CreI, I-CsmI, F-TevI, F-TevII,I-TevI, I-TevII, I-AniI, I-CvuI, I-LlaI, I-NanI, I-MsoI, I-NitI, I-NjaI,I-PakI, I-PorI, I-PpoI, I-ScaI, I-SceI, I-Ssp6803I, PI-PkoI, PI-PkoII,PI-PspI, PI-TfuI, PI-TliI.

Homing endonucleases which are especially preferably used are thosewhich are found as such naturally, especially preferably those which arefound naturally in organelles. Most preferably, the homing endonucleasesoriginate from organisms which live at similar temperatures to plants.Those which are of particular interest in this contest are the homingendonucleases identified in yeast and Chlamydomonas species. Naturally,it is also feasible to utilize homing endonucleases which are isolatedfrom extremophilic organisms, as long as they are active in the plastidsof the plant to be transformed.

The following are very especially preferred:

-   -   I-CeuI (Cote M J and Turmel M (1995) Curr Genet 27:177-183.;        Gauthier A et al. (1991) Curr Genet 19:43-47; Marshall (1991)        Gene 104:241-245; GenBank Acc. No.: Z17234 nucleotides 5102 to        5758),    -   I-ChuI (Cote V et al. (1993) Gene 129:69-76; GenBank Acc. No.:        L06107, nucleotides 419 to 1075),    -   I-CmoeI (Drouin M et al. (2000) Nucl Acids Res 28: 4566-4572),    -   I-CpaI from Chiamydomonas pallidostigmatica (GenBank Acc. No.:        L36830, nucleotides 357 to 815; Turmel M et al. (1995) Nucleic        Acids Res 23:2519-2525; Turmel, M et al. (1995) Mol Biol Evol        12:533-545; see also SEQ ID NO: 13 and 14)    -   I-CpaII (Turmel M et al. (1995) Mol Biol Evol 12:533-545;        GenBank Acc. No.: L39865, nucleotides 719 to 1423),    -   I-CreI (Wang J et al. (1997) Nucleic Acids Res 25: 3767-3776;        Dürrenberger, F and Rochaix J D (1991) EMBO J. 10:3495-3501;        GenBank Acc. No.: X01977, nucleotides 571 to 1062),    -   I-CsmI (Ma D P et al. (1992) Plant Mol Biol 18:1001-1004)    -   I-NanI (Elde M et al. (1999) Eur J. Biochem. 259:281-288;        GenBank Acc. No.: X78280, nucleotides 418 to 1155),    -   I-NitI (GenBank Acc. No.: X78277, nucleotides 426 to 1163),    -   I-NjaI (GenBank Acc. No.: X78279, nucleotides 416 to 1153),    -   I-PpoI encodes on the extrachromosomal DNA in the nucleus of        Physarum polycephalum (Muscarella D E and Vogt V M (1989) Cell        56:443-454; Lin J and Vogt V M (1998) Mol Cell Biol        18:5809-5817; GenBank Acc. No.: M38131, nucleotides 86 to 577).        In addition, the longer sequence encoding I-PpoI, which        originates from an alternative start codon, may also be used.        This sequence comprises the nucleotides 20 to 577 in the        sequence of GenBank Acc. No. M38131. It is preferred to use the        shorter sequence; however, it can be substituted at any location        by the corresponding, longer one. A sequence which is especially        preferred for the purposes of the present invention is an        artificial sequence which encodes for the same amino acid as the        sequence of the nucleotides 86 to 577 of the sequence of GenBank        Acc. No.: M38131 (see also SEQ ID NO: 5, 11, 12, 70 or 71),    -   I-PspI (GenBank Acc. No.: U00707, nucleotides 1839 to 3449),    -   I-ScaI (Monteilhet C et al. (2000) Nucleic Acids Res 28:        1245-1251; GenBank Acc. No.: X95974, nucleotides 55 to 465)    -   I-SceI from the mitochondria of bakers' yeast (WO 96/14408; U.S.        Pat. No. 5,962,327 Seq ID NO: 1 therein),    -   Endo SceI (Kawasaki et al. (1991) J Biol Chem 266:5342-5347,        identical with F-SceI; GenBank Acc. No.: M63839, nucleotides 159        to 1589),    -   I-SceII (Sarguiel B et al. (1990) Nucleic Acids Res        18:5659-5665),    -   I-SceIII (Sarguiel B et al. (1991) Mol Gen Genet. 255:340-341),    -   I-Ssp68031 (GenBank Acc. No.: D64003, nucleotides 35372 to        35824),    -   I-TevI (Chu et al. (1990) Proc Natl Acad Sci USA 87:3574-3578;        Bell-Pedersen et al. (1990) Nucleic Acids Res 18:3763-3770;        GenBank Acc. No.: AF158101, nucleotides 144431 to 143694),    -   I-TevII (Bell-Pedersen et al. (1990) Nucleic Acids Res        18:3763-3770; GenBank Acc. No.: AF158101, nucleotides 45612 to        44836),    -   I-TevIII (Eddy et al. (1991) Genes Dev. 5:1032-1041),

Commercially available homing endonucleases such as I-CeuI, I-SceI,I-PpoI, PI-PspI or PI-SceI are very especially preferred. Most preferredare I-SceI and I-PpoI. While the gene encoding I-PpoI can be used in itsnatural form, the gene encoding I-SceII contains an editing locus.Since, in contrast to yeast mitochondria, the editing in question is notperformed in the plastids of higher plants, an artificial sequence whichencodes the I-SceI protein must be employed for the heterologousexpression of this enzyme (U.S. Pat. No. 5,866,361).

In addition to the above-stated homing endonucleases, there are furtherintron-encoded enzymes which can be found at homologous locations of thegenomes of related organisms. As a rule, these homing endonucleases havesimilar sequence specificity and are therefore equally suited as DSBIenzyme for the introduction of a DSB into the plastome at the DSBrecognition sequence. The corresponding sequence is thus also recognizedby Sob2593c, Clu2593c, Col2593c, Ciy2593c, Hla2593c, Cag2593c, I-CvuI,I-PakI, Tmu2593c, Msp2593c, I-MsoI, Sdu2593c, Mvi2593m, Nol2593m orAca2593m, in addition to I-CreI. Corresponding sequence is alsorecognized by I-CecI, I-CmoI, I-CelI, I-CpaIII, I-CmuI, I-CluI, I-SobIor I-AstI, in addition to I-CeuI. A corresponding sequence is alsorecognized by Cbr1931c, Cfr1931c, Cme1931c, Cge1931c, Pcr1931c,Msp1931c, Mso1931C, Ptu1931c, Cvu1931m, Msp1931m, Msol931m, Nol1931m,Acal931m or Sne1931b, in addition to I-CpaI. Moreover, introns existwhich are inserted at position 1951 of the 23S rDNA (numbering refers tohomologous position in the 23S rDNA of E. coli). These introns, again,encode putative homing endonucleases which can be used as. DSBI enzymesfor the specific cleavage of the plastidic DNA. They include, forexample, Cbr1951c, Msp1951c, Mso1951c, Cvu1951m or Acal951m (Lucas P etal. (2001) Nucl Acids Res 29:960-969).

Most preferred are the homing endonucleases of the protein sequencesdescribed by SEQ ID NO: 5, 12 or 14. When preparing correspondingexpression cassettes, accordingly, nucleic acid sequences are employedwhich encode a protein as shown in SEQ ID NO: 5, 12 and 14,respectively; especially preferred in this context is the use of thenucleic acid sequences as shown in SEQ ID NO: 11 and 13 or of anexpression cassette as shown in SEQ ID NO: 4.

The enzymes can be isolated from their source organisms in the mannerwith which the skilled worker is familiar and/or the nucleic acidsequence encoding them can be cloned. The sequences of a variety ofenzymes have been deposited at GenBank (see above).

Examples of artificial DSBI enzymes by way of example are chimericnucleases which are composed of an unspecific nuclease domain and asequence-specific DNA binding domain (for example consisting of zincfingers) (Smith J et al. (2000) Nucl Acids Res 28(17):3361-3369;Bibikova M et al. (2001) Mol Cell Biol. 21:289-297). Thus, for example,the catalytic domain of the restriction endonuclease FokI has been fusedwith zinc finger binding domains, whereby the specificity of theendonuclease has been defined (Chandrasegaran S & Smith J (1999) BiolChem 380:841-848; Kim Y G & Chandrasegaran S (1994) Proc Natl Acad SciUSA 91:883-887; Kim Y G et al. (1996) Proc Natl Acad Sci USA93:1156-1160). The catalytic domain of the yeast Ho endonuclease, too,has already been conferred a predefined specificity, using theabove-described technique, by fusing it with the zinc finger domain oftranscription factors (Nahon E & Raveh D (1998) Nucl Acids Res26:1233-1239).

As mentioned, zinc finger proteins are particularly suitable as DNAbinding domain for the purpose of chimeric nucleases. These DNA-bindingzinc finger domains can be adapted to match any desired DNA sequence.Suitable methods for the preparation of such zinc finger domains aredescribed and known to the skilled worker (Beerli R R et al. (2000) ProcNatl Acad Sci USA 97(4):1495-1500; Beerli R R et al. (2000) J Biol Chem275(42):32617-32627; Segal D J and Barbas C F 3rd. (2000) Curr Opin ChemBiol 4(1):34-39; Kang J S and Kim J S (2000) J Biol Chem275(12):8742-8748; Beerli R R et al. (1998) Proc Natl Acad Sci USA95(25):14628-14633; Kim J S et al. (1997) Proc Natl Acad Sci USA94(8):3616-3620; Klug A (1999) J Mol Biol 293(2):215-218; Tsai S Y etal. (1998) Adv Drug Deliv Rev 30(1-3):23-31; Mapp A K et al. (2000) ProcNatl Acad Sci USA 97(8):3930-3935; Sharrocks A D et al. (1997) Int JBiochem Cell Biol 29(12):1371-1387; Zhang L et al. (2000) J Biol Chem275(43):33850-33860). Methods for the preparation and selection of zincfinger DNA binding domains with high sequence specificity have beendescribed (WO 96/06166, WO 98/53059, WO 98/53057). Fusing a DNA bindingdomain thus obtained with the catalytic domain of an endonuclease (suchas, for example, the FokI or Ho endonuclease) allows the preparation ofchimeric nucleases with any desired specificity which can be employedadvantageously as DSBI enzymes for the purposes of the presentinvention.

Artificial DSBI enzymes with modified sequence specificity can also beprepared by mutating known restriction endonucleases or homingendonucleases by methods known to the skilled worker. The mutagenesis ofmaturases with the purpose of obtaining a modified substrate specificityis of particular interest, besides the mutagenesis of homingendonucleases. Frequently, maturases share many features with homingendonucleases and, if appropriate, they can be converted into nucleasesby carrying out few mutations. This has been shown, for example, for thematurase in the bakers' yeast bi2 intron. Only two mutations in thematurase-encoding open reading frame (ORF) sufficed to confer a homingendonuclease activity to this enzyme (Szczepanek & Lazowska (1996) EMBOJ 15:3758-3767).

Further artificial nucleases can be generated with the aid of mobilegroup II introns and the proteins encoded by them, or parts of theseproteins. Many mobile group II introns, together with the proteinsencoded by them, form RNA-protein particles which are capable ofrecognizing, and cleaving, DNA in a sequence-specific manner. Here, thesequence specificity can be adapted to suit the needs by mutatingcertain intron regions (see hereinbelow) (WO 97/10362).

The skilled worker is familiar with various methods for introducing aDSBI enzyme into plastids or expressing it therein.

The following may be mentioned by way of example, but not by limitation:

-   a) Nuclear expression using plastidic transit peptides    -   An expression cassette encoding a DSBI enzyme fusion protein can        be constructed in the manner known to the skilled worker,        introduced into the nucleus and—optionally—integrated stably        into the chromosomal DNA. For transport into the plastids, the        DSBI enzyme is preferably expressed in fusion with a plastid        localization sequence (PLS). Methods for the direct        transportation, into the plastids, of proteins which per se are        not localized in the plastids, and a variety of PLS sequences,        have been described (Klosgen R B and Weil J H (1991) Mol Gen        Genet 225(2):297-304; Van Breusegem F et al. (1998) Plant Mol        Biol 38(3):491-496). Preferred are those PLS which, after        translocation of the DSBI enzyme into the plastids, are cleaved        enzymatically from the DSBI enzyme moiety. Especially preferred        is the PLS which is derived from the plastidic Nicotiana tabacum        transketolase or from another transit peptide (for example the        transit peptide of the small Rubisco subunit or of the        ferredoxin NADP oxidoreductase, and also        isopentenyl-pyrophosphate isomerase-2) or its functional        equivalent. Promoters which are suitable for expression in the        nucleus are, in principle, all those which make possible an        expression in plants. Examples can be found further below.        Preferred are constitutive promoters such as the CaMV 35S        promoter or the nitrilase-1 promoter of the Arabidopsis nitl        gene (GenBank Acc. No.: Y07648.2, nucleotides 2456 to 4340;        Hillebrand H et al. (1998) Plant Mol Biol 36 (1):89-99;        Hillebrand H et al. (1996) Gene 170(2):197-200).

Preferred PLS Sequences Are:

-   -   i) the Arabidopsis isopentenyl isomerase (IPP) transit peptide        (GenBank Acc. No.: NC 003074; nucleotides 604657-604486)    -   ii) transit peptides derived from the small subunit (SSU) of        ribulose-bisphosphate carboxylase (Rubisco ssu) from, for        example, pea, maize, sunflower or Arabidopsis.        -   Arabidopsis thaliana: GenBank Acc. No.: for example            AY054581, AY054552;        -   pea, GenBank Acc. No.: for example X00806, nucleotides 1086            to 1256; X04334, X04333 (Hand JM (1989) EMBO J            8(11):3195-206). Especially preferred in this context are:            expression cassette and transit peptide (pea, rbcS3A) from            vector pJIT117 (Guerineau F (1988) Nucleic Acids Res            16(23):11380. Especially preferred is the peptide sequence            as shown in SEQ ID NO: 35. Most preferred for the use in            constructing suitable expression constructs is the nucleic            acid sequence as shown in SEQ ID NO: 34.        -   maize, GenBank Acc. No.: for example S42568, S42508        -   sunflower, GenBank Acc. No.: Y00431, nucleotides 301 to 465.    -   iii) transit peptides derived from plant fat biosynthesis genes,        such as the transit peptide of the plastid acyl carrier protein        (ACP) (for example the Arabidopsis thaliana beta-ketoacyl-ACP        synthetase 2; GenBank Acc. No.: AF318307), stearyl-ACP        desaturase, β-ketoacyl-ACP synthase or acyl-ACP thioesterase        (for example A. thaliana mRNA for acyl-(acyl carrier        protein)thioesterase: GenBank Acc. No.: Z36911).    -   iv) the GBSSI (starch granule bound synthase I) transit peptide    -   v) the transit peptide of the LHCP II genes.    -   Especially preferred is the PLS of the plastidic tobacco        transketolase (SEQ ID NO: 36). To express corresponding fusion        proteins, different PLS nucleic acid cassettes can be used in        the three reading frames as KpnI/BamHI fragments for the        purposes of the present invention (the translation start (ATG        codon) is localized in the NcoI cleavage site) (pTP09 SEQ ID NO:        37; pTP10 SEQ ID NO: 38; pTP11 SEQ ID NO: 39).    -   A further example of a PLS to be employed advantageously is the        transit peptide of the plastidic Arabidopsis thaliana        isopentenyl-pyrophosphate isomerase-2 (IPP-2) (SEQ ID NO: 40).        The nucleic acid sequences encoding three cassettes        (corresponding to the three reading frames) of the PLS from the        Arabidopsis thaliana isopentenyl-pyrophosphate isomerase-2        (IPP-2) can be used very especially preferably (EcoRV/SalI        cassettes with ATG; IPP-9 SEQ ID NO: 41; ipp-10 SEQ ID NO: 42;        IPP-11 SEQ ID NO: 43).    -   The nucleic acids according to the invention can be of synthetic        origin or have been obtained naturally or comprise a mixture of        synthetic and natural nucleic acid components, or else consist        of various heterologous gene segments from a variety of        organisms.    -   The sequence encoding the transit peptide can comprise all or        part of the peptide sequence of the original protein. An        accurate determination of the amino acid residues which are        essential for the transport is not required as long as the        functionality of the PLS—which is the transport into the        plastid—is ensured and the function of the DSBI enzyme is not        entirely destroyed. Very especially preferred are the following        PLS sequences:    -   PLS1: N-MASSSSLTLSQAILSRSVPRHGSASSSQLSPSSLTFSGLKSNPNITTSRRR        TPSSAAAAAVVRSPAIRASAATETIEKTETAGS-C (SEQ ID NO: 36). Corresponds        to the PLS of the tobacco plastidic transketolase.    -   PLS2: N-MSASSLFNLPLIRLRSLALSSSFSSFRFAHRPLSSISPRKLPNFRAFSGTA        MTDTKDGSRVDM-C (SEQ ID NO: 40). Corresponds to the PLS of        isopentenyl-pyrophosphate isomerase-2 (IPP-2), the last        methionine preferably being the start methionine of the DSBI        enzyme.    -   The homing endonuclease as shown in SEQ ID NO: 69 is a preferred        fusion protein of the native I-Ppo I nuclease and the IPP        plastid localization sequence. This protein is preferably        encoded by the sequence with the SEQ ID NO: 68.    -   For the purposes of the present invention, fusion proteins of        PLS and DSBI enzyme come under the term “DSBI enzyme”. If a DSBI        enzyme is expressed in the nucleus, the DSBI enzyme is        preferably understood as meaning a fusion protein of PLS and        DSBI enzyme.    -   The invention furthermore relates to fusion proteins of DSBI        enzymes with plastid localization sequence (PLS), sequences and        expression cassettes comprising a fusion protein of a plastid        localization sequence (PLS) and a DSBI enzyme under the control        of a promoter which is functional in the plant nucleus. Such        suitable promoters are known to the skilled worker and described        further below. The expression cassette can comprise further        elements such as, for example, transcription terminators and/or        selection markers.

-   b) Expression in plastids    -   An expression in plastids can also take place by the direct        introduction of an expression cassette for the DSBI enzyme into        plastids, if appropriate integration into the plastidic DNA, and        expression of the DSBI enzyme. In a preferred embodiment, this        expression cassette is present in the transformation construct        which comprises the insertion sequence.    -   Promoters which can be employed are, firstly, specific plastid        or chromoplast promotors as detailed hereinbelow. However, a        directed expression in plastids can also be achieved by using        for example a viral, bacterial or bacteriophage promoter,        introducing the resulting expression cassette into the plastidic        DNA, and then inducing expression by the corresponding viral,        bacterial or bacteriophage RNA polymerase. The corresponding RNA        polymerase, in turn, can be introduced into the plastids in        various ways, preferably by nuclear transformation in the form        of a fusion protein with a PLS. Suitable methods have been        described (WO 95/16783, WO 97/06250, U.S. Pat. No. 5,925,806).        It is preferably introduced into plastids by microinjection,        especially preferably by means of particle bombardment.

-   c) Introduction in the form of RNA    -   The DSBI enzyme can also be introduced into plastids by        introducing the mRNA—for example mRNA which has been generated        in vitro—which encodes the DSBI enzyme via, for example,        microinjection, particle bombardment (biolistic methods), or        polyethylene glycol- or liposome-mediated transfection. This        embodiment is advantageous since no DSBI-enzyme-encoding        sequences remain in the plastome or genome in this case.        Preferably, the RNA encoding the DSBI enzyme is generated by        in-vitro transcription in the manner with which the skilled        worker is familiar.

-   d) Introduction in the form of the protein    -   The DSBI enzyme can be introduced into plastids directly, for        example via microinjection, particle bombardment (biolistic        methods) or polyethylene glycol transfection or        liposome-mediated transfection. This embodiment is advantageous        since no DSBI-enzyme-encoding sequences remain in the plastome        or genome. Such a method is described, for example, by Segal D J        et al. (1995) Proc Natl Acad Sci USA 92:806-810.    -   The DSBI enzyme can be introduced into plant cells as a fusion        protein with the VirE2 or VirF protein of an agrobacterium and a        PLS. Such methods have been described for example for Cre        recombinase (Vergunst AC et al. (2000) Science 290:979-982).        This embodiment is advantageous since no DSBI enzyme-encoding        sequences remain in the genome.

Of course, combinations of the above-described possibilities are alsofeasible.

The expression cassette for the DSBI enzyme is preferably present on theinsertion sequence or on the transformation construct.

The DSBI enzyme is preferably introduced, or activated, simultaneouslywith, or after, the introduction of the insertion sequence into theplastids. Expression and/or activation at the correct site and thecorrect point in time can be ensured by various approaches:

-   a) Inducible expression    -   The expression of a DSBI enzyme can be controlled using an        inducible promoter, preferably a chemically inducible promoter.        To this end, for example, the expression cassette which encodes        the DSBI enzyme can be transformed stably into the plastidic or        nuclear DNA of a master plant. If it is transformed into the        nuclear genome, the subcellular localization must be ensured—as        described above—by suitable PLS transit peptides. Shortly before        or during the transformation with the insertion sequence or the        transformation construct, the expression of the DSBI enzyme will        then be switched on by applying a suitable inductor, which        depends on the choice of the inducible system. The skilled        worker is familiar with a variety of methods or promoters for        induced expression. Chemical compounds or else physical stimuli        such as, for example, increased temperature or wounding and the        like can act as stimulus. Various examples are described further        below.-   b) Inducible activity    -   The DSBI enzyme can already exist in the plastids of the master        plant when the activity is induced by suitable techniques at the        selected point in time only, for example by addition of chemical        compounds. Such methods have been described for        sequence-specific recombinases (Angrand P O et al. (1998) Nucl        Acids Res 26(13):3263-3269; Logie C and Stewart A F (1995) Proc        Natl Acad Sci USA 92(13):5940-5944; Imai T et al. (2001) Proc        Natl Acad Sci USA 98(1):224-228). Fusion proteins of the DSBI        enzyme and the ligand binding domain of a steroid hormone        receptor (for example the human androgen receptor, or mutated        variants of the human estrogen receptor as described therein)        are employed in these methods. Induction can be effected with        ligands such as, for example, estradiol, dexamethasone,        4-hydroxytamoxifen or raloxifen.    -   Some of the DSBI enzymes are active in the form of the dimer        (homo- or heterodimer) (1-CreI forms a homodimer; I-PpoI forms a        homodimer, Flick K E et al. (1998) Nature 394: 96-101). In        general, enzymes of the LAGLIDADG family tend to form homodimers        when only one LAGLIDADG motif is present per monomer (Jurica M S        & Stoddard B L (1999) Cell Mol Life Sci 55:1304-1326; I-CeuI may        be mentioned by way of example). A dimerization can be designed        to be inducible, for example by substituting the natural        dimerization domains by the binding domain of a        low-molecular-weight ligand. Addition of a dimeric ligand then        brings about the dimerization of the fusion protein. Such        inducible dimerization methods are described, as is the        preparation of the dimeric ligands (Amara J F et al. (1997) Proc        Natl Acad Sci USA 94(20): 10618-10623; Muthuswamy S K et        al. (1999) Mol Cell Biol 19(10): 6845-6857; Schultz L W and        Clardy J (1998) Bioorg Med Chem Lett 8(1):1-6; Keenan T et        al. (1998) Bioorg Med. Chem. 6(8):1309-1335).-   c) Cotransfection    -   The expression cassette encoding the DSBI enzyme is preferably        introduced into the plastids simultaneously with the insertion        sequence. In this context, the expression cassette for the DSBI        enzyme and the insertion sequence may be present on one DNA        molecule or else on two separate DNA molecules. Preferably, the        two sequences are present together on one DNA molecule, so that        the expression cassette is present in the transformation        construct comprising the insertion sequence.    -   In an especially preferred embodiment, the sequence encoding the        DSBI enzyme is removed from the genome of the transformed        plasmids after homoplastomic plants have been regenerated. The        skilled worker is familiar with a variety of methods for doing        so which are detailed further below.

Some of the above-described DSBI enzymes (in particular homingendonucleases) can have recognition sequences in the intermediate hostE. coli, which is preferably used. Since, moreover, some expressioncassettes for expression in plastids are also functional in E. coli, itis preferred to prevent expression of the DSBI enzyme in E. coli invarious ways with which the skilled worker is familiar in order to avoidany disadvantageous effects on E. coli during amplification of theexpression cassette. Thus, for example, several consecutive codons whichoccur rarely in E. coli (for example the codons AGA and AGG, whichencode arginine) can be inserted into the reading frame of the DSBIenzyme. This prevents expression in E. coli, but—owing to the differentcodon usage—continues to make possible expression in the plastids. As analternative, promoters which are not active in E. coli, but are activein the plastids of higher plants can be used (for example promoters ofthe nuclear encoded plastidic RNA polymerases [NEP promoters; seehereinbelow]). A preferred method is the use of promoters which arerecognized neither by the plastids nor by E. coli (for example viralpromoters or bacteriophage promoters) and which only become functionalby the simultaneous presence of the corresponding viral/bacteriophageRNA polymerase. Such methods are known to the skilled worker anddescribed hereinbelow. Furthermore, it is feasible to destroy therelevant DSB recognition sequences in E. coli or to use a different hostwhich has no DSB recognition sequences for the DSBI enzyme in question.Moreover, it is feasible and advantageous to have the coding region ofthe DSBI enzyme in promoterless form for amplification in E. coli. Inthis case, the sequence which encodes the DSBI enzyme is preferablypresent on a plasmid which is capable of integration into the plastidicgenome of the plant to be transformed. Here, the integration site can bechosen in such a way that the gene encoding the DSBI enzyme comes underthe control of a promoter which is naturally present in the plastome orhas been inserted artificially into the plastome, thus resulting inexpression of the DSBI enzyme in the plastids. A further preferredembodiment ensures that the gene encoding the DSBI enzyme can later bedeleted from the plastome (see hereinbelow). In addition, it is possibleto create a linkage between a promoter and a DSBI enzyme by adding sucha promoter in vitro upstream of the open reading frame by means of PCRtechniques with which the skilled worker is familiar. The PCR productcan then be used for introduction into the plant plastids. Moreover,nonfunctional parts of an expression cassette for a DSBI enzyme can begenerated and amplified in E. coli when these parts undergorecombination with one another after introduction into plant plastids(for example by means of homologous recombination in overlapping regionsof the nonfunctional moieties of the expression cassette), thus givingrise to a functional expression cassette.

“Recognition sequence for the directed induction of DNA double-strandbreaks” (hereinbelow “DSB recognition sequence” for double-strand breakrecognition sequence) generally refers to those sequences which permitrecognition and cleavage by a DSBI enzyme under the conditions in theplastids of the plant cell or plant used in each case. Especiallypreferred are DSB recognition sequences for-homing endonucleases whichare encoded naturally in mitochondria or the nucleus of other organisms.Also, it is possible to use DSB recognition sequences of homingendonucleases which are derived from plastids (for example from greenalgae). Preferably, the DSB recognition sequence is singular in theplastidic DNA, i.e. a double-strand break is only generated at thelocation thus predefined. However, cases where more than one DSBrecognition sequence is present in the plastome are also feasible. Thisis the case in particular when the DSB recognition sequence is localizedin duplicated genes (for example in inverted repeats). In the lattercase, more than one identical DSB recognition sequence exist, but theircontext is identical, so that, again, directed insertion takes place.Indeed, it is preferred that integration into all copies takes place,which also requires cleavage in all copies. DSB recognition sequenceswhich, while occurring more than once in one plastome, are localized inthe same plastomic context (for example in repeats or in geneduplications) come under the term “singular DSB recognition sequences”for the purposes of the present invention.

Preferably, the plant employed, or the cell derived therefrom, ispredominantly homoplastomic or homotransplastomic with regard to the DSBrecognition sequence, i.e. the predominant number of the plastidic DNAmolecules present in the plastid contain this DSB recognition sequence.For the purposes of the present invention, such plants are also referredto as master plants.

In principle, two types of DSB recognition sequences can be used:

-   a) Natural, endogenous DSB recognition sequences    -   As has been demonstrated within the scope of the present        invention, the plastomes of higher plants comprise various        sequences which can act as recognition sequences for DSBI        enzymes (for example homing endonucleases), even though no such        endonucleases have been demonstrated in higher plants to date.        Such DSB recognition sequences can be identified by screening        the plastidic DNA sequence using the known DSB recognition        sequences (for example those described in Table 2). The        plastidic genome of various plants is known        (http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.        html). The sequences of the plastomes of the following have been        reported:        -   Arabidopsis thaliana (Sato S et al. (1999) DNA Res. 6            (5):283-290) (GenBank Acc. No.: AP000423; NCBI Acc. No.            NC_(—)000932)        -   Epifagus virginiana (Beechdrops; Wolfe K H et al. (1992) J            Mol Evol 35(4):304-317; NCBI Acc. No.: NC_(—)001568; GenBank            Acc. No.: M81884)        -   Lotus japonicus (Kato T et al. (2000) DNA Res 7(6):323-330;            NCBI Acc. No.: NC_(—)002694; GenBank Acc. No.:AP002983)        -   Oryza sativa (rice) (Hiratsuka J et al. (1989) Mol Gen Genet            217(2-3):185-194; NCBI Acc. No.: NC_(—)001320; GenBank Acc.            No: X15901),        -   Marchantia polymorpha (Liverwort; Ohyama K et al. (1988) J            Mol Biol 203(2):281-298; Yamano Y et al. (1984) Nucl Acids            Res 12(11):4621-4624; GenBank Acc. No.: X04465 and Y00686;            NCBI Acc. No.: NC_(—)001319)        -   Nicotiana tabacum (tobacco) (GenBank Acc. No.: Z00044 and            S54304; NCBI Acc. No.: NC_(—)001879; Shinozaki K et            al. (1986) EMBO J 5:2043-2049)        -   Oenothera elata ssp. hookeri (Monterey evening primrose;            GenBank Acc. No.: AJ271079; NCBI Acc. No.: NC_(—)002693;            Hupfer H et al. (2000) Mol Gen Genet 263(4):581-585)        -   Medicago truncatula (Gen Bank Acc. No.: AC093544)        -   Pinus thunbergii (black pine; Tsudzuki J et al. (1994) Curr            Genet 26(2):153-158; NCBI Acc. No.:NC_(—)001631; GenBank            Acc. No.: D17510)        -   Spinacia oleracea (GenBank Acc. No.: AJ400848 J01442 M12028            M16873 M16878 M27308 M55297 X00795 X00797 X01724 X04131            X04185 X05916 X06871)        -   Triticum aestivum (wheat; GenBank Acc. No.: AB042240; NCBI            Acc. No.: NC_(—)002762) and        -   Zea mays (GenBank Acc. No.: X86563; NCBI Acc. No.:            NC_(—)001666)    -   In addition, further plastomes can be sequenced in order to        identify DSB recognition sites therein. In general, it suffices        to isolate highly-conserved regions from the plastome by PCR        methods with which the skilled worker is familiar and to        sequence these regions only.    -   Furthermore, it is possible to determine natural, endogenous DSB        recognition sites experimentally, for example by isolating the        plastidic DNA (for example by the method of Mariac P et        al. (2000) BioTechniques 28:110-113), amplifying the plastidic        genome fragments to be taken into consideration by means of PCR        or by using synthetic fragments and carrying out a restriction        analysis with the DSBI enzyme in question. This restriction        analysis is preferably carried out under conditions as they        prevail in the plastid of a higher plant.    -   Moreover, the endogenous DSB recognition sequences for natural        homing endonucleases which have been identified and described in        Table 1 within the scope of the present invention are located in        the conserved regions of the plastome so that—in particular        taking into consideration the given variability, with regard to        their respective recognition sequences, of the homing        endonucleases mentioned in each case—it can be assumed that        these recognition sequences are found virtually universally in        all the plastomes of higher plants. The positions shown in Table        1 reveal in each case the sequence stated and the        reverse-complementary sequence, since all of the recognition        regions shown in Table 1 are localized in the inverted repeat of        the plastidic genome. Homing endonucleases which are especially        preferred among those mentioned in Table 1 are I-CpaI, I-CeuI,        I-ChuI, I-CpaII and I-CreI.    -   The recognition sequences identified thus can be used for the        insertion of foreign DNA by generating a double-strand break by        introducing the corresponding DSBI enzyme. If the DSB        recognition sequence were to be located in a highly-conserved        region within a gene of the organelle genome, the foreign DNA is        preferably inserted in the form of a self-splicing intron, which        allows the reconstitution of the mRNA of the affected gene (see        hereinbelow).    -   The skilled worker is furthermore familiar with methods in which        any endogenous sequence can act as recognition sequence for        chimeric, mutated or artificial endonucleases, by subjecting        their DNA recognition region to directed modification, for        example by modification of a zinc finger domain fused to an        endonuclease domain, or by modification of the RNA sequence of a        group II intron RNA/protein complex (see hereinabove; WO        96/06166, Bibikova M et al. (2001) Mol Cell Biol 21:289-297).

Published DSB Sequence Position in the Position in the DSBI recognitionin the tobacco plastome wheat plastome enzyme sequence plastome Acc.Z00044 Acc. AB042240 I-DmoI ATGCGCGCCGGAACT GTGCGGGTCGGAACTc(108281-108310) 118010-118039 TACCCGGCAAGGCAT TACCCGACAAGGAAT134316-134345 c(96855-96884) I-CpaI CGATCCTAAGGTAGC CGGTCCTAAGGTAGC108263-108285 96837-96859 GAAATTCA GAAATTCC c(134341-134363)c(118035-118057) I-CeuI CGTAACTATAACGGT CGTAACTATAACGGT c(134346-134374)c(118040-118068) CCTAAGGTAGCGAA CCTAAGGTAGCGAA 108252-108280 96826-96854I-ChuI GAAGGTTTGGCACCT GAAGGTTTGGCACCT 108832-108861 97405-97434CGATGTCGGCTCATC CGATGTCGGCTCTTC c(133765-133794) c(117460-117489)I-CpaII CCCGGCTAACTCTGT ATCGGCTAACTCTGT c(139398-139417)c(123374-123393) GCCAG GCCAG 103209-103228 91501-91520 I-CreICTGGGTTCAAAACGT CTGGGTTCAGAACGT 108925-108954 97498-97527CGTGAGACAGTTTGG CGTGAGACAGTTCGG c(133672-133701) c(117367-117396) I-SceITACCCTGTTATCCCT CAGCCTGTTATCCCTA c(108804-108781) c(97377-97354)AGCGTAACT GAGTAACT 133822-133845 117516-117540 Position in the Positionin the Position in the DSBI rice plastome maize plastome Arabidopsisplastome enzyme Acc. X159019 Acc. X86563 Acc. AP000423 I-DmoI117846-117875 121617-121646 131977-132006 c(97243-97272)c(101091-101120) c(106643-106672) I-CpaI 97224-97246 101073-101095106625-106647 c(117871-117893) c(121642-121664) c(132002-132024) I-CeuIc(117876-117904) 101062-101090 c(132007-132035) 97214-97242c(121647-121675) 106614-106642 I-ChuI 97792-97821 101641-101670107194-107223 c(117297-117326) c(121067-121096 c(131426-131455) I-CpaIIc(123351-123370) c(127108-127127) c(137169-137188) 91748-9176795610-95629 101461-101480 I-CreI 97885-97914 deviating 107287-107316c(117204-117233) sequence: c(131333-131362) ctgggttcagaacgtcgtgagacgttcgg c(120975-121003) 101734-101762 I-SceI c(97741-97764)c(101590-101613) c(107143-107166) 117354-117377 121124-121147131483-131506

Tab 1: Preferred endogenous cleavage sites in the plastidic genomes oftobacco, wheat, rice, maize and Arabidopsis. c=complementary. Acc. No:GenBank Accession Number (http://www.ncbi.nlm.nih.gov/).

-   -   Singular cleavage sites of restriction endonucleases also exist        in the plastidic genome. However, they are usually located in        less highly-conserved regions and can therefore not necessarily        be exploited universally in all plant species. The following may        be mentioned by way of example:        -   a) With the sequence GGCCTTTATGGCC the enzyme SfiI has a            singular recognition site in the plastidic genome of            Arabidopsis (GenBank Acc. No.: AP000423) at position            40846-40858.        -   b) In the plastidic genome of maize (GenBank Acc. No.:            X86563), there is a singular cleavage site for the enzyme            AscI at position 42130-42137, with the sequence GGCGCGCC.        -   c) In the plastidic genome of rice (GenBank Acc. No.:            X159019), there is a singular cleavage site for the enzyme            SgfI at position 77309-77316, with the sequence GCGATCGC,            and for the enzyme AscI at position 39776-39783 with the            sequence GGCGCGCC.        -   d) In the plastidic genome of tobacco (Accession Z00044),            there is in each case a singular cleavage site for the            enzyme SfiI at position 42475-42487, with the sequence            GGCCTTTATGGCC, for the enzyme SgrI at position 78522-78529,            with the sequence CACCGGCG, and for the enzyme PmeI at            position 120895-120902, with the sequence GTTTAAAC.        -   e) In the plastidic genome of wheat (Accession AB042240),            there is in each case a singular cleavage site for the            enzyme PmeI at position 59331-59338, with the sequence            GTTTAAAC, a singular cleavage site for the enzymes NarI,            KanI, EheI and BbeI at position 41438-41443, with the            recognition sequence GGCGCC, and a recognition region for            the enzyme SfiI at position 112656-112668, with the sequence            GGCCCAGGGGGCC.    -   All these plants with endogenous, natural DSB recognition        sequences constitute, in a manner of speaking, naturally        occurring master plants. In them, the DSB recognition sequence        is naturally present in homoplastomic form. This eliminates the        need for the introduction and selection of artificial DSB        recognition sequences.

-   b) Artificially Introduced DSB Recognition Sequences    -   The skilled worker realizes that the recognition region for a        rare enzyme introduced into a master plant need not be of        natural origin. In principle, any recognition sequence of any        DSBI enzyme can be inserted at any position of the plastidic        DNA. The preparation is preferably carried out using a construct        for inserting the DSB recognition sequence (hereinbelow DSBR        construct). Preferably, the DSBR construct comprises a selection        marker to facilitate the selection of transplastomic plants with        the successfully inserted DSB recognition sequence, which        selection is required for generating suitable master plants. The        skilled worker is familiar with a variety of selection markers        which make possible selection of plastids (see hereinbelow).        aada, nptII or BADH are preferred, with aadA being especially        preferred. Selection is carried out for example with the aid of        the “segregation and sorting” process, with which the skilled        worker is familiar (described by way of example in Example 4).        The selection marker is preferably constructed in such a way        that a subsequent deletion from the plastome is made possible.        Such methods are known to the skilled worker and described        hereinbelow.    -   Thus, it is preferred first to generate a plant which is        homotransplastomic with regard to the inserted DSB recognition        sequence and which has a DSB recognition sequence in all or the        predominant number of the plastids of the plant in question.        Such plants can advantageously be employed as master plants.    -   In addition to the selection marker, the DSBR construct may        comprise further sequences. These may contain for example        further regulatory elements for the expression of the insertion        sequences to be introduced subsequently. In a preferred        embodiment, the selection marker introduced within the construct        for insertion of the DSB recognition sequence is deleted after        obtaining the homoplastomic master plant by methods known to the        skilled worker (see hereinbelow).    -   In a preferred embodiment, the DSBR construct comprises, for        making possible a site-specific insertion, further flanking        sequences at least one, preferably at both sides of the DSB        recognition sequence, which flanking sequences have sufficient        length and homology with corresponding target sequences in the        plastome to ensure site-specific insertion by means of        homologous recombination.

Owing to the large number of the DSBI enzymes with defined recognitionsequences which have been described in the prior art, it is possible,and preferred, to generate master plants which have a plurality ofdifferent singular DSB recognition sequences incorporated into theirplastidic genome.

The recognition sequences for the respective DSBI enzymes listed arementioned hereinbelow in Table 2 by way of example, but not bylimitation.

TABLE 2 Recognition sequences and source organism of the DSBI enzymes(“{circumflex over ( )}” shows the cleavage site of the DSBI enzymewithin a recognition sequence.) DSBI Source enzyme organism Recognitionsequence I-AniI Aspergillus 5′-TTGAGGAGGTT{circumflex over( )}TCTCTGTAAATAANNNNNNNNNNNNNNN nidulans3′-AACTCCTCCAAAGAGACATTTATTNNNNNNNNNNNNNNN{circumflex over ( )} I-CvuIChlorella 5′-CTGGGTTCAAAACGTCGTGA{circumflex over ( )}GACAGTTTGGvulgaris 3′-GACCCAAGTTTTGCAG{circumflex over ( )}CACTCTGTCAAACC I-CsmIChlamydomonas 5′-GTACTAGCATGGGGTCAAATGTCTTTCTGG smithii I-CmoeIChlamydomonas 5′-TCGTAGCAGCT{circumflex over ( )}CACGGTT moewusii3′-AGCATCG{circumflex over ( )}TCGAGTGCCAA I-CreI Chlamydomonas5′-CTGGGTTCAAAACGTCGTGA{circumflex over ( )}GACAGTTTGG reinhardtii3′-GACCCAAGTTTTGCAG{circumflex over ( )}CACTCTGTCAAACC I-ChuIChlamydomonas 5′-GAAGGTTTGGCACCTCG{circumflex over ( )}ATGTCGGCTCATChumicola 3′-CTTCCAAACCGTG{circumflex over ( )}GAGCTACAGCCGAGTA I-CpaIChlamydomonas 5′-CGATCCTAAGGTAGCGAA{circumflex over ( )}ATTCA pallido-3′-GCTAGGATTCCATC{circumflex over ( )}GCTTTAAGT stigmatica I-CpaIIChlamydomonas 5′-CCCGGCTAACTC{circumflex over ( )}TGTGCCAG pallido-3′-GGGCCGAT{circumflex over ( )}TGAGACACGGTC stigmatica I-CeuIChlamydomonas 5′-CGTAACTATAACGGTCCTAA{circumflex over ( )}GGTAGCGAAeugametos 3′-GCATTGATATTGCCAG{circumflex over ( )}GATTCCATCGCTT I-DmoIDesulfuro- 5′-ATGCCTTGCCGGGTAA{circumflex over ( )}GTTCCGGCGCGCAT coccus3′-TACGGAACGGCC{circumflex over ( )}CATTCAAGGCCGCGCGTA mobilis I-SceISaccharomyces 5′-AGTTACGCTAGGGATAA{circumflex over ( )}CAGGGTAATATAGcerevisiae 3′-TCAATGCGATCCC{circumflex over ( )}TATTGTCCCATTATATC5′-TAGGGATAA{circumflex over ( )}CAGGGTAAT 3′-ATCCC{circumflex over( )}TATTGTCCCATTA (“Core” sequence) I-SceII Saccharomyces5′-TTTTGATTCTTTGGTCACCC{circumflex over ( )}TGAAGTATA cerevisiae3′-AAAACTAAGAAACCAG{circumflex over ( )}TGGGACTTCATAT I-SceIIISaccharomyces 5′-ATTGGAGGTTTTGGTAAC{circumflex over ( )}TATTTATTACCcerevisiae 3′-TAACCTCCAAAACC{circumflex over ( )}ATTGATAAATAATGG I-SceIVSaccharomyces 5′-TCTTTTCTCTTGATTA{circumflex over ( )}GCCCTAATCTACGcerevisiae 3′-AGAAAAGAGAAC{circumflex over ( )}TAATCGGGATTAGATGC I-SceVSaccharomyces 5′-AATAATTTTCT{circumflex over ( )}TCTTAGTAATGCCcerevisiae 3′-TTATTAAAAGAAGAATCATTA{circumflex over ( )}CGG I-SceVISaccharomyces 5′-GTTATTTAATG{circumflex over ( )}TTTTAGTAGTTGGcerevisiae 3′-CAATAAATTACAAAATCATCA{circumflex over ( )}ACC I-SceVIISaccharomyces 5′-TGTCACATTGAGGTGCACTAGTTATTAC cerevisiae PI-SceISaccharomyces 5′-ATCTATGTCGGGTGC{circumflex over ( )}GGAGAAAGAGGTAATcerevisiae 3′-TAGATACAGCC{circumflex over ( )}CACGCCTCTTTCTCCATTA F-SceISaccharomyces 5′-GATGCTGTAGGC{circumflex over ( )}ATAGGCTTGGTTcerevisiae 3′-CTACGACA{circumflex over ( )}TCCGTATCCGAACCAA F-SceIISaccharomyces 5′-CTTTCCGCAACA{circumflex over ( )}GTAAAATT cerevisiae3′-GAAAGGCG{circumflex over ( )}TTGTCATTTTAA I-LlaI Lactococcus5′-CACATCCATAAC{circumflex over ( )}CATATCATTTTT lactis3′-GTGTAGGTATTGGTATAGTAA{circumflex over ( )}AAA I-MsoI Monomastix5′-CTGGGTTCAAAACGTCGTGA{circumflex over ( )}GACAGTTTGG species3′-GACCCAAGTTTTGCAG{circumflex over ( )}CACTCTGTCAAACC I-NanI Naegleria5′-AAGTCTGGTGCCA{circumflex over ( )}GCACCCGC andersoni3′-TTCAGACC{circumflex over ( )}ACGGTCGTGGGCG I-NitI Naegleria5′-AAGTCTGGTGCCA{circumflex over ( )}GCACCCGC italica3′-TTCAGACC{circumflex over ( )}ACGGTCGTGGGCG I-NjaI Naegleria5′-AAGTCTGGTGCCA{circumflex over ( )}GCACCCGC jamiesoni3′-TTCAGACC{circumflex over ( )}ACGGTCGTGGGCG I-PakI Pseudendo-5′-CTGGGTTCAAAACGTCGTGA{circumflex over ( )}GACAGTTTGG clonium3′-GACCCAAGTTTTGCAG{circumflex over ( )}CACTCTGTCAAACC akinetum I-PorIPyrobaculum 5′-GCGAGCCCGTAAGGGT{circumflex over ( )}GTGTACGGGorganotrophum 3′-CGCTCGGGCATT{circumflex over ( )}CCCACACATGCCC I-PpoIPhysarum 5′-TAACTATGACTCTCTTAA{circumflex over ( )}GGTAGCCAAATpolycephalum 3′-ATTGATACTGAGAG{circumflex over ( )}AATTCCATCGGTTTA “Coresequence”:       CTCTCTTAA{circumflex over ( )}GGTAGC      GAGAG{circumflex over ( )}AATTCCATCG I-ScaI Saccharomyces5′-TGTCACATTGAGGTGCACT{circumflex over ( )}AGTTATTAC capensis3′-ACAGTGTAACTCCAC{circumflex over ( )}GTGATCAATAATG I-Ssp6803ISynechocystis 5′-GTCGGGCT{circumflex over ( )}CATAACCCGAA species3′-CAGCCCGAGTA{circumflex over ( )}TTGGGCTT PI-PfuI Pyrococcus5′-GAAGATGGGAGGAGGG{circumflex over ( )}ACCGGACTCAACTT furiosus Vc13′-CTTCTACCCTCC{circumflex over ( )}TCCCTGGCCTGAGTTGAA PI-PfuIIPyrococcus 5′-ACGAATCCATGTGGAGA{circumflex over ( )}AGAGCCTCTATAfuriosus Vc1 3′-TGCTTAGGTACAC{circumflex over ( )}CTCTTCTCGGAGATATPI-PkoI Pyrococcus 5′-GATTTTAGAT{circumflex over ( )}CCCTGTACCkodakaraensis 3′-CTAAAA{circumflex over ( )}TCTAGGGACATGG KOD1 PI-PkoIIPyrococcus 5′-CAGTACTACG{circumflex over ( )}GTTAC kodakaraensis3′-GTCATG{circumflex over ( )}ATGCCAATG KOD1 PI-PspI Pyrococcus5′-AAAATCCTGGCAAACAGCTATTAT{circumflex over ( )}GGGTAT sp.3′-TTTTAGGACCGTTTGTCGAT{circumflex over ( )}AATACCCATA PI-TfuIThermococcus 5′-TAGATTTTAGGT{circumflex over ( )}CGCTATATCCTTCCfumicolans 3′-ATCTAAAA{circumflex over ( )}TCCAGCGATATAGGAAGG ST557PI-TfuII Thermococcus 5′-TAYGCNGAYACN{circumflex over ( )}GACGGYTTYTfumicolans 3′-ATRCGNCT{circumflex over ( )}RTGNCTGCCRAARA ST557 PI-ThyIThermococcus 5′-TAYGCNGAYACN{circumflex over ( )}GACGGYTTYT hydro-3′-ATRCGNCT{circumflex over ( )}RTGNCTGCCRAARA thermalis PI-TliIThermococcus 5′-TAYGCNGAYACNGACGG{circumflex over ( )}YTTYT litoralis3′-ATRCGNCTRTGNC{circumflex over ( )}TGCCRAARA PI-TliII Thermococcus5′-AAATTGCTTGCAAACAGCTATTACGGCTAT litoralis I-TevI Bacteriophage5′-AGTGGTATCAAC{circumflex over ( )}GCTCAGTAGATG T43′-TCACCATAGT{circumflex over ( )}TGCGAGTCATCTAC I-TevII Bacteriophage5′-GCTTATGAGTATGAAGTGAACACGT{circumflex over ( )}TATTC T43′-CGAATACTCATACTTCACTTGTG{circumflex over ( )}CAATAAG F-TevIBacteriophage 5′-GAAACACAAGA{circumflex over( )}AATGTTTAGTAAANNNNNNNNNNNNNN T43′-CTTTGTGTTCTTTACAAATCATTTNNNNNNNNNNNNNN{circumflex over ( )} F-TevIIBacteriophage 5′-TTTAATCCTCGCTTC{circumflex over ( )}AGATATGGCAACTG T43′-AAATTAGGAGCGA{circumflex over ( )}AGTCTATACCGTTGAC

Also comprised are deviations (degenerations) of the recognitionsequence which nevertheless continue to make possible recognition andcleavage by the DSBI enzyme in question. Such deviations—also inconnection with different framework conditions such as, for example,calcium or magnesium concentrations—have been described (Argast G M etal. (1998) J Mol Biol 280: 345-353). Furthermore comprised are coresequences of these recognition sequences. It is known that the innerportions of the recognition sequences also suffice for an induceddouble-strand break and that the outer portions are not necessarilyrelevant, but may have an effect on the cleavage efficiency. Thus, forexample, an 18 bp core sequence can be defined for I-SceI. The term “DSBrecognition sequence” thus also comprises all essentially identicalrecognition sequences. Essentially identical recognition sequencesrefers to those recognition sequences which, while deviating from therecognition sequence identified as being optimal for the enzyme inquestion, still permit cleavage by the same.

Various localization sites (in the case of already existing endogenousDSB recognition sequences) or integration sites (in the case ofartificially generated DSB recognition sequences) are possible for theDSB recognition sequence. Examples which may be mentioned are:

-   a) Localization (integration)-in a transcriptionally silent region    -   Localization (integration) of the DSB recognition sequence in a        transcriptionally silent region of the plastidic genome        (intergenic region) is the preferred embodiment. In this manner,        an adverse effect on the plastids' functions can be largely        ruled out. In this context, it must be noted that, if        appropriate, suitable regulatory elements such as promoters and        the like must also be introduced for expression to take place.)-   b) Localization (integration) in a transcriptionally active but    noncoding (intercistronic) region    -   The advantage of this localization (integration) is that the        insertion sequence to be introduced is thereby ultimately        encoded in a plastidic operon and promoter(s)/terminator(s) need        not be introduced separately, but those present endogenously at        this locus can, but do not have to, be utilized. In such a case,        only ribosome binding sites should be present at a suitable        distance upstream of the coding region of the foreign genes to        be introduced.    -   However, it is also feasible that an intergenic region is not        entirely transcriptionally silent, for example because        transcriptional termination from an adjacent gene or operon is        only inefficient.)-   c) Localization (integration) in a transcriptionally active coding    region.    -   The localization (integration) described under a) and b) of the        DSB recognition sequence at a noncoding locus has the advantage        that the insertion of the foreign DNA is highly likely not to        affect the function of the plastidic genome. However, noncoding        regions are less well conserved than coding regions. In order to        have available as universal a method as possible which works in        many plant species, the DSB recognition sequence (and therefore        the insertion sequence) is, in an especially preferred        embodiment, localized in the coding sequence of an existing        gene. Destruction of the gene function by introducing the DSB        recognition sequence (in the case of an artificially generated        DSB recognition sequence), or the introduction of the insertion        sequence, is prevented inventively, in a preferred variant of        this embodiment, by introducing the DSB recognition sequence, or        the insertion sequence, within an intron. In this manner, the        complete coding mRNA is regenerated at the site of integration        by splicing the pre-RNA of the gene.

DSB recognition sequences which do not occur naturally in the plastidicDNA can be introduced into the plastidic DNA in various ways. Exampleswhich may be mentioned are:

-   a) Integration by means of double crossover    -   Integration into the plastidic genome is preferably carried out        with the aid of the above-described methods with which the        skilled worker is generally familiar (double crossover).-   b) Integration using natural, endogenous DSB recognition sequences-   c) Integration using recombinases and corresponding recognition    sequences.

Even though the procedure for inserting an artifical DSB recognitionsequence into the plastidic DNA is relatively complicated and, in casea), corresponds to the plastid transformation method currently describedin the prior art, this complicated procedure only has to be carried outonce. The resulting homotransplastomic master plant can then be employedfor any number of different subsequent transformations using the methodaccording to the invention, which makes possible a substantial increasein the transformation efficiency: instead of having to carry out theconventional selection process for a homotransplastomic plant everysingle time, it only has to be carried out once in the present context.

“Deactivation of the functionality” of a DSB recognition sequence meansthat, owing to insertion of the insertion sequence at or near theposition of the double-strand break, the DSB recognition sequence isdestroyed, i.e. the corresponding DSBI enzyme no longer recognizes theregion and, accordingly, no longer induces a double-strand break at thisposition.

Construction of the transformation construct with the insertion sequence

Using one of the above-described master plants or cells derived fromthem which contain a natural and/or an artificially generated DSBrecognition sequence in the plastome, the insertion sequence is insertedinto said DSB recognition sequence within a transformation process. Thisis effected with the simultaneous presence of a DSBI enzyme, whichrecognizes one of the DSB recognition sequences in the plastome.

In its simplest form, the transformation construct consists only of theinsertion sequence itself, for example of an expression cassette whichis to ensure the expression of a certain gene in the plastids. Thesequence-specific induction of double-strand breaks suffices to ensurethat this insertion sequence is placed at this position and thus tobring about the deactivation of the DSB recognition sequence.

In a preferred embodiment, the insertion sequence comprises at least onenucleic acid sequence to be expressed. To ensure expression(transcription and/or translation), they are to be provided withregulatory elements, depending on the embodiment and the insertion site.If insertion takes place at a transcriptionally active locus, nopromoter sequences are required, as described above. The sequences to beexpressed are advantageously provided in any case with ribosome bindingsites at a suitable distance upstream of the open reading frame, or arealready equipped naturally with such sites. These regulatory sequencesor parts thereof can, however, also be present naturally in the plastomeor introduced into the plastidic DNA together with the DSB recognitionsequence as early as in the first step, i.e. in the generation of anonnatural master plant.

An increase of the insertion efficiency and insertion accuracy can bebrought about by flanking the insertion sequence present in thetransformation construct and the DSB recognition sequence by homologoussequence regions which, owing to the induced double-strand break, ensurehomologous recombination. In a preferred embodiment, the insertionsequence comprises flanking homology sequences A′ and B′, the sequenceto be introduced into the plastidic DNA being located between A′ and B′.The DSB recognition sequence is flanked by homology sequences A and B,respectively, the DSB recognition sequence being located between A andB. A and B can be of natural origin or have been introduced in contextwith the insertion of nonnatural DSB recognition sequences. A and A′ andB and B′, respectively, are sufficiently long and sufficientlyhomologous to one another to ensure a homologous recombination between Aand A′, and B and B′, respectively.

In a further embodiment, the DSB recognition sequence is flanked merelyby a homologous sequence A which has sufficient homology to a sequenceA′ which flanks the insertion sequence unilaterally.

With regard to the homology sequences, “sufficient length” preferablymeans sequences with a length of at least 20 base pairs, preferably atleast 50 base pairs, especially preferably at least 100 base pairs, veryespecially preferably at least 250 base pairs, most preferably at least500 base pairs.

With regard to the homology sequences A and A′, and B and B′,respectively, “sufficient homology” preferably means sequences whichhave at least 70%, preferably 80%, by preference at least 90%,especially preferably at least 95%, very especially preferably at least99%, most preferably 100% homology within these homology sequences overa length of at least 20 base pairs, preferably at least 50 base pairs,especially preferably at least 100 base pairs, very especiallypreferably at least 250 base pairs, most preferably at least 500 basepairs.

Homology between two nucleic acids is understood as meaning the identityof the nucleic acid sequence over in each case the entire sequencelength which is calculated by alignment with the aid of the programalgorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin,Genetics Computer Group (GCG), Madison, USA), setting the followingparameters:

-   -   Gap Weight: 12 Length Weight: 4    -   Average Match: 2,912 Average Mismatch: −2,003

Since homologous recombination is promoted by the induced double-strandbreak, the requirements regarding length and homology of the sequencesare markedly less than is the case for example in the case ofconventional homologous recombination. In this context, the homologousregions can also be markedly smaller than 250 bp. The advantage of usinghomology sequences is that, when A′ and B′ are different, or when onlyone homology sequence A′ is being used, a directed insertion of theinsertion sequence into the plastidic DNA can take place.

The transformation construct or the insertion sequence preferablycomprises a selection marker which makes possible the selection oftransplastomic plastids (see hereinbelow), especially preferably aada,BADH or a binding-type marker. The selection marker is preferablyconstructed in such a way that subsequent deletion from the plastome ismade possible. Such methods are known to the skilled worker anddescribed hereinbelow.

The insertion sequence or the transformation construct preferably hasthe structure and sequence of an intron. As a rule, the naturallyoccurring introns are modified in such a manner for this purpose thatthey meet the requirements of the method according to the invention.Such artificial introns are especially preferred when they are to beinserted into a transcriptionally active or even coding region, forexample, using a natural, endogenous DSB recognition sequence.Preferably, insertion takes place in such a way that the insertedsequence is removed completely by splicing the pre-mRNA. The RNA whichhas been spliced out (that is to say the artificial intron) nowconstitutes the mRNA, for example for the translation of proteinsencoded on it. This method has further advantages:

-   -   The introns utilized show pronounced secondary folding so that a        relatively stable RNA results. The genes of interest which are        encoded in the intron can therefore be expressed at a        particularly high level, as has been demonstrated, for example,        in E. coli (Chan K Y W et al. (1988) Gene 73:295-304).    -   When the intron is integrated into a gene, the transcription of        the intron is subject to the regulatory control of the gene into        which the intron has been integrated. This is why all regulatory        elements upstream or downstream of the gene(s) of interest can        be dispensed with in the intron. The constructs can thus be kept        correspondingly small, and it is certain that transcription does        indeed work, including in the species under investigation. The        utilization of heterologous regulatory elements involves the        residual risk that these elements are not functional in the        investigated plastids of the plant species in question. The        utilization of homologous sequences can, owing to the sequence        duplication, lead to spontaneous recombination events with the        endogenous sequences and thus to instability of the organelle        genome. Owing to the possibility of largely being able to        dispense with the introduction of regulatory elements—for        example by encoding the gene of interest in an intron which is        inserted into a transcriptionally active plastome region—many        other disadvantages of conventional plastid transformation can        be avoided with the method according to the invention in this        embodiment, in addition to increasing the insertion and        distribution ability of the transformation constructs.

Moreover, all introns can be used when the relevant factors whichmediate splicing are simultaneously expressed in the plastids orimported into them. Preferably, the splicing factors are encoded in theintron itself. Group II introns, which themselves encode at least one ofthe splice factors, are especially preferred in this embodiment. Theyinclude the Lactococcus Ll.ltrB intron. Likewise preferred introns arethose which naturally occur in the plastids of higher plants, especiallygroup II introns, very especially preferably introns which encode aprotein, most preferably introns of the trnK genes of the plastidicgenome. In the latter case, the introns from the trnk genes of theplastids from the species Arabidopsis, maize and tobacco are especiallypreferred.

Preferred introns are those which have a self-splicing activity whichdoes not depend on further protein factors, or introns which utilizegeneral factors for splicing which are universally present, andtherefore also in plastids, and also introns which themselves encodefactors required for splicing. These introns include, for example,

-   a) the group I intron from Tetrahymena (GenBank Acc. No.: X54512;    Kruger K et al. (1982) Cell 31:147-157; Roman J and Woodson S    A (1998) Proc Natl Acad Sci USA 95:2134-2139)-   b) the group II rIl intron from Scenedesmus obliquus (GenBank Acc.    No.: X17375.2 nucleotides 28831 to 29438; Holländer V and Kück    U (1999) Nucl Acids Res 27: 2339-2344; Herdenberger F et al. (1994)    Nucl Acids Res 22: 2869-2875; Kück U et al. (1990) Nucl Acids Res    18:2691-2697).-   c) the Ll.LtrB intron (GenBank Acc. No.: U50902 nucleotides 2854 to    5345)-   d) the Arabidopsis trnK intron (GenBank Acc. No.: AP000423,    complementary nucleotides 1752 to 4310)-   e) the maize trnK intron (GenBank Acc. No.: X86563, complementary    nucleotides 1421 to 3909)-   f) the tobacco trnK intron (GenBank Acc. No.: Z00044, complementary    nucleotides 1752 to 4310).

Not only heterologous introns, but also introns which naturally occur inthe plastids of the plant in question can be utilized. Heterologousintrons—for example heterologous trnk introns—are preferred to avoidinstabilities brought about by sequence duplication. In a preferredembodiment, introns which occur naturally in the plastids of the plantin question are modified in such a way that they have a sequencehomology of less than 95%, preferably 80%, especially preferably 70%with the sequence of the starting intron, while still being able toretain their function.

In a further preferred embodiment, a factor which brings about splicingof the intron in question is available in trans, i.e. it is not encodedin the intron itself. If this factor is not naturally present in theplastid in question, but first has to be introduced into it, such aprocedure can be effected in various ways with which the skilled workeris familiar. Examples which may be mentioned are the introduction of asuitable coding sequence, which is capable of expression, into theplastome or the introduction into the nuclear DNA; in the latter case,the factor is preferably fused with a PLS.

Especially preferred introns are those which naturally encode a DSBenzyme (in particular a homing endonuclease). Especially preferred isthe intron Cp.LSU2 from Chlamydomonas pallidostigmatica, which encodesthe enzyme I-CpaI (Turmel M et al. (1995) Mol Biol Evol 12:533-545).Also preferred are the group-II introns from yeast mitochondria.

In a preferred embodiment, the intron sequence is adapted to suit theinsert site so that they can splice at this locus. In the case of groupI introns, this adaptation can relate to the internal guide sequence(IGS) and in the case of the group II introns the exon binding sequence(EBS) I and/or II.

In the case of the maize trnK intron, it must be noted that the proteinencoded by the trnK intron, which also comprises the maturase function,is probably not functional in its naturally encoded form withoutediting. It has been demonstrated that editing (His420Tyr) of thecorresponding mRNA takes place in barley plastids (Vogel J et al. (1997)J Mol Biol 270:179-187). Tyrosine at position 420 of the matK protein ishighly conserved. In the monocots rice and maize, too, a codon encodingHis has been found at the corresponding position in the coding DNA. Itcan therefore be assumed that the matK transcript is also edited inthose plants, as is the case in barley. Since, however, other plantspecies may, if appropriate, not be able to provide such RNA editing, apreferred embodiment provides that the matK gene in the maize trnkintron is already modified at DNA level by a suitable His/Tyrsubstitution, so that RNA editing is no longer required. For example,the sequence CATTATCATAGTGGAT of the maize trnk intron can be mutatedinto CATTATTATAGTGGAT.

In the case of group I introns, the splicing site is determined by thepairing of IGS with the exon of the corresponding transcript, which exonis located 5′ and/or 3′ relative to the intron (Lambowitz A M & BelfortM (1993) Annu Rev Biochem 62:587-622). Using techniques which are knownto the skilled worker, such as PCR or the synthetic generation ofnucleotide sequences, the IGS can be matched to any group I introns insuch a way that splicing takes place at the predefined insertion sitewithin the DSB recognition region. The modified IGS is designed in sucha way that it can undergo—at least partial—base pairing with thesequences of the transcript 5′ and 3′ of the insertion site. The C.pallidostigmatica CpLSU2 intron, which encodes the homing endonucleaseI-CpaI, is preferably utilized. If this intron is utilized in connectionwith the expression of the DSBI enzyme I-CpaI, whereby insertion of theDNA to be transformed into the 23S rDNA of the plastidic genome ofhigher plants results, no adaptation of the intron is necessary.Insertion takes place at a locus in the plastidic genome of higherplants which is homologous to the locus at which the intron is naturallypresent in C. pallidostigmatica. This intron is therefore alreadydesigned in such a way that pairings with the 5′ and 3′ exon can beundergone and that correct splicing in this nucleotide environment takesplace. Furthermore preferred is the group I intron from Tetrahymenathermophila, where, as a 413 bp intervening sequence (IVS), itinterrupts the 26S rRNA coding region (Accession V01416 J01235nucleotides 53 to 465). The IGS with the sequence 5′-ggaggg-3′ which canbe found naturally (Waring R B et al. 1985 Cell 40: 371-380; Been, M D &Cech, T R 1986 Cell 47: 207-216) can be adapted to the new insertionsite by techniques with which the skilled worker is familiar. If, forexample, integration into the DSB recognition site of the I-CpaI enzymeat the position identified by ^ (cggtcct^aaggagcgaaattc) is desired, themutated, adapted IGS can, for example, have the following sequence:5′-gggacc-3′.

In group II introns, which are mobile, further activities in addition tomaturase are frequently encoded in the protein moiety of theribonucleoprotein complex. However, these are not necessarily requiredfor the method described and can therefore be deleted. Indeed, deletionis preferred since it makes the construct in question smaller and easierto handle. The skilled worker is familiar with a variety of options forremoving such activities from the protein moiety. For example, this canbe effected by generating a synthetic gene which comprises only thedesired regions, or by suitable PCR methods.

Self-splicing group II introns have a conserved structure and generallyconsist of 6 different domains. Domain I comprises the exon bindingsites (EBS1 and EBS2) which, during the splicing procedure, interactwith the exon located 5′ from the intron. In addition, an interactionbetween the “δ region” (located immediately 5′ of EBS1) and the “δ′region” at the 3′ exon takes place (Lambowitz A M & Belfort M (1993)Annu Rev Biochem 62:587-622; Michel F & Ferat J L (1995) Annu RevBiochem 64:435-461). These sequences can be adapted by techniques withwhich the skilled worker is familiar, such as synthetic generation ofthe introns or suitable PCR methods, in each case in such a way thatcorrect choice of the splicing sites at the insert site chosen in theDSB recognition region is ensured. This is done in such a way that theregions mentioned are modified so that base pairings with thecorresponding sequences upstream (intron binding sequences, IBS) anddownstream (δ′) of the artificial insertion sequence can be undergone.If, for example, cggtcctaaggt^agcgaaattc is chosen as insertion site (^)for the Ll.LtrB intron in the I-CpaI recognition region, the δ regionand the EBS1 region can, for example, adopt the sequence TCGCTACCTTAG(natural sequence: TTATGGTTGTG), and EBS2 for example the sequence GACCG(natural sequence: ATGTG). If the Arabidopsis thaliana trnK intron isselected, the δ region and the EBS1 region can, for example, adopt thesequence CGCTACCTTAGG (natural sequence: AATGTTAAAAA), assuming the sameinsertion site as indicated for the Ll.LtrB intron.

If the DSB recognition sequence takes the form of a natural, endogenousrecognition sequence of a homing endonuclease, a selected intron ispreferably inserted at the site of the DSB recognition region at whichthe intron belonging to the homing endonuclease in question can also befound naturally.

The artificial insertion site of an intron in the DSB recognition siteis preferably chosen such that 5′ and 3′ of the intron inserted as manybases as possible correspond to those of the natural insertion site ofthe intron in question and that the DSB recognition sequence is nolonger functional after insertion of the intron. Very especiallypreferably, the nucleotide located in each case immediately upstream ordownstream of the insert site of the intron corresponds to that at thenatural insertion site.

In an especially preferred embodiment, the intron is flanked by homologysequences in order to make possible a directed insertion. Here, thehomology sequences are—as described above—homologous to the sequencesflanking the DSB recognition sequence and thus make possible an accurateinsertion.

The invention therefore furthermore also relates to DNA constructscomprising at least one nucleic acid and intron sequence elements whichare capable of ensuring, in a ribonucleic acid sequence derived fromsaid DNA construct, the deletion of the ribonucleic acid fragmentencoding said nucleic acid sequence, where said nucleic acid sequence isheterologous with regard to said intron sequence elements.

In a preferred embodiment, the nucleic acid sequence is flanked at leastby a splice acceptor sequence and a splice donor sequence.

In a further embodiment, the DNA construct comprises, at the 5′ and the3′ end, sequences H1 and H2, respectively, which have sufficient lengthand homology with plastid sequences H1′ and H2′, respectively, to ensurehomologous recombination between H1 and H1′, and H2 and H2′,respectively, and thus insertion of the H1- and H2-flanked sequence intothe plastome.

The invention furthermore relates to a transgenic plastidic DNAcomprising at least one nucleic acid sequence and intron sequenceelements which are capable of ensuring, in a ribonucleic acid sequencederived from said transgenic plastidic DNA, the deletion of saidribonucleic acid fragment encoding said nucleic acid sequence, wheresaid nucleic acid sequence is heterologous with regard to said intronsequence elements. In a preferred embodiment, the nucleic acid sequenceis flanked by at least one splice acceptor sequence and one splice donorsequence.

To construct a transformation vector, the insertion sequence or thetransformation construct can be cloned into a standard vector such aspBluescript or pUC18. In a preferred embodiment, the insertion sequenceor the transformation construct is applied as a linear or linearized DNAmolecule.

Preferably, only the portion of transformation vector which comprisesthe insertion sequence or the transformation construct with, ifappropriate, homology sequences, selection marker and/or the expressioncassette for the DSBI enzyme is applied. If all or some of the homologysequences are dispensed with, the linearized DNA molecule is preferablyobtained by digestion with restriction endonucleases which generatesingle-stranded DNA overhangs at one or at both ends which arecompatible with those generated by the DSBI enzyme in the plastidic DNA.

In a preferred embodiment, the transformation vector can compriseelements (for example a plastidic ORI (origin of replication)), whichmake it possible for the vector autonomously to replicate in the plastidor stably to exist in the plastids as extrachromosomal DNA molecule,before being integrated into the plastidic DNA. Such methods are knownto the skilled worker (U.S. Pat. No. 5,693,507; U.S. Pat. No. 5,932,479;WO 99/10513). This method is preferred since it increases the copynumber of the insertion sequences which is available for integration inthe plastid.

One of the above-described constructs can be introduced into theplastids of a suitable master plant using one of the methods described.Microinjection is preferred, particle bombardment is particularlypreferred.

Cloning, Expression, Selection and Transformation Methods

“Expression cassette” means—for example regarding the expressioncassette for the DSBI enzyme—those constructions in which the DNA to beexpressed is in operable linkage with at least one genetic controlelement which makes possible or regulates its expression (i.e.transcription and/or translation). In this context, expression can be,for example, stable or transient, constitutive or inducible. A varietyof direct methods (for example transfection, particle bombardment,microinjection) or indirect methods (for example agrobacterialinfection, viral infection) stated hereinbelow is available to theskilled worker for the introduction, and these methods will be statedhereinbelow.

An operable linkage is generally understood as meaning an arrangement inwhich a genetic control sequence can exert its function with regard to anucleic acid sequence, for example encoding a DSBI enzyme. Function, inthis context, can mean for example the control of the expression, i.e.transcription and/or translation, of the nucleic acid sequence, forexample encoding a DSBI enzyme. Control, in this context, comprises forexample initiating, increasing, governing or suppressing the expression,i.e. transcription and, if appropriate, translation. Governing, in turn,can be tissue and/or timing-specific. It may also be inducible, forexample by certain chemicals, stress, pathogens and the like.

An operable linkage is understood as meaning for example the sequentialarrangement of a promoter, of the nucleic acid sequence to beexpressed—for example encoding a DSBI enzyme—and, if appropriate,further regulatory elements such as, for example, a terminator in such away that each of the regulatory elements can fulfill its function uponexpression of the nucleic acid sequence, for example encoding a DSBIenzyme. In this context, operable linkage need not necessarily exist onthe transformation constructs themselves. Operable linkage can alsoresult as a consequence of the insertion into the nuclear or plastidicDNA, where the regulatory elements are already present in the nuclear orplastidic DNA. In this respect, the regulatory elements can be naturallypresent or else introduced in a preceding step, for example whenintroducing an artificial DSB recognition sequence.

Direct linkage in the chemical sense is not necessarily required in thiscontext. Genetic control sequences such as, for example, enhancersequences, can also exert their function on the target sequence frompositions which are further away, or indeed from other DNA molecules.Preferred arrangements are those in which the nucleic acid sequence tobe expressed—for example encoding a DSBI enzyme—is positioned behind asequence which acts as promoter, so that the two sequences are bondedcovalently with one another. Preferably, the distance between thepromoter sequence and the nucleic acid sequence—for example encoding aDSBI enzyme—is less than 200 base pairs, especially preferably less than100 base pairs, very especially preferably less than 50 base pairs.

The skilled worker is familiar with a variety of routes to obtain one ofthe transformation constructs according to the invention, vectorscomprising them or one of the expression cassettes. They can be preparedby means of customary recombination and cloning techniques as aredescribed for example in Maniatis T, Fritsch E F and Sambrook J,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989) and in Silhavy T J, Berman M L andEnquist L W, Experiments with Gene Fusions, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel F M et al.,Current Protocols in Molecular Biology, Greene Publishing Assoc. andWiley Interscience (1987). The direct fusion of a nucleic acid sequencewhich acts as promoter and a nucleotide sequence to be expressed—forexample encoding a DSBI enzyme—is preferred.

The term “genetic control sequences” is to be understood in the broadsense and refers to all those sequences which influence the generationor the function of an expression cassette or transformation vector.Genetic control sequences ensure transcription and, if appropriate,translation in the nucleus (or cytoplasm) or plastids. Preferably, theexpression cassettes according to the invention comprise a promoter 5′upstream of the respective nucleic acid sequence to be expressed and aterminator sequence as additional genetic control sequence 3′downstream, and, if appropriate, further customary regulatory elements,in each case in operable linkage with the nucleic acid sequence to beexpressed.

Genetic control sequences are described, for example, by “Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)” or “Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnolgy, CRC Press, Boca Raton, Fla., eds.:Glick and Thompson, Chapter 7, 89-108” and the references cited therein.

Examples of such control sequences are sequences to which the inductorsor repressors bind and thus regulate the expression of nucleic acid. Thenatural regulation of these sequences may still be present before theactual structural genes, in addition to these novel control sequences orinstead of these sequences, and, if appropriate, can have beengenetically modified so that the natural regulation has been switchedoff and gene expression enhanced. However, the expression cassette canalso be simpler in structure, that is to say no additional regulatorysignals are inserted before the abovementioned genes and the naturalpromoter together with its regulation is not removed. Instead, thenatural control sequence is mutated in such a way that regulation nolonger takes place and gene expression is enhanced. These modifiedpromoters can also be placed by themselves before the natural genes inorder to increase the activity.

Depending on the host organism or the starting organism described ingreater detail hereinbelow, which is converted into a geneticallymodified or transgenic organism by the introduction of the expressioncassettes or vectors, different control sequences are suitable.

Promoters which are suitable for nuclear expression (for example of aviral/bacteriophage RNA polymerase or of a DSBI enzyme with plastidictransit peptide) are, in principle, all those which are capable ofgoverning the expression of genes, in particular foreign genes, inplants.

Suitable promoters are those which make possible constitutive expressionin plants (Benfey et al. (1989) EMBO J. 8:2195-2202). In particular, aplant promoter or a promoter derived from a plant virus is used bypreference. Especially preferred is the promoter of the cauliflowermosaic virus 35S transcript (Franck et al. (1980) Cell 21:285-294; Odellet al. (1985) Nature 313:810-812; Shewmaker et al. (1985) Virology140:281-288; Gardner et al. 1986, Plant Mol. Biol. 6, 221-228) or the19S CaMV promoter (U.S. Pat. No. 5,352,605 and WO 84/02913). As isknown, this promoter comprises different recognition sequences fortranscriptional effectors which, in their totality, lead to largelypermanent and constitutive expression of the gene introduced (Benfey etal. (1989) EMBO J 8:2195-2202). A further suitable constitutive promoteris the “Rubisco small subunit (SSU)” promotor (U.S. Pat. No. 4,962,028).A further example of a suitable promoter is the leguminB promoter(GenBank Acc. No.: X03677). Examples of further preferred constitutivepromoters are the Agrobacterium nopaline synthase promoter, the TR dualpromoter, the Agrobacterium OCS (octopine synthase) promoter, theubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649),the promoters of the vacuolar ATPase subunits, the FBPaseP promoter (WO98/18940) or the promoter of a proline-rich protein from wheat (WO91/13991). Other suitable constitutive promoters which are preferred forthe purposes of the present invention are the Super promoter (Ni M etal. (1995) Plant J 7:661-676; U.S. Pat. No. 5,955,646) and thenitrilase-1 promoter of the Arabidopsis nit1 gene (GenBankAcc. No.:Y07648.2, nucleotides 2456 to 4340; Hillebrand H et al. (1998) Plant MolBiol 36 (1):89-99; Hillebrand H et al. (1996) Gene 170(2):197-200).

Promoters which are preferred are inducible promoters, especiallypreferably chemically inducible promoters (Aoyama T and Chua N H (1997)Plant J 11:605-612; Caddick M X et al. (1998) Nat. Biotechnol16:177-180; Review: Gatz (1997) Annu Rev Plant Physiol Plant Mol Biol48:89-108) by means of which expression can be controlled at aparticular point in time. Examples which may be mentioned are the PRP1promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), asalicylic-acid-inducible promoter (WO 95/19443), abenzenesulfonamide-inducible promoter (EP-A-0388186), atetracyclin-inducible promoter (Gatz et al. (1992) Plant J 2:397-404),an abscisic-acid-inducible promoter (EP-A 335 528), an ethanol-induciblepromoter (Salter MG et al. (1998) Plant J. 16:127-132), theheavy-metal-inducible metallothionein I promoter (Amini S et al. (1986)Mol Cell Biol 6:2305-2316), the steroid-inducible MMTV LTR promoter(Izant J G et al. (1985) Science 229:345-352) and acyclohexanone-inducible promoter (WO 93/21334). Especially preferred isthe inducible expression of a PLS/DSBI enzyme fusion protein in thenucleus. Inducible promoters also comprise those which are capable ofregulation by certain repressor proteins (for example tet, lac). Suchrepressor proteins can be translocated into the plastids in fusion withPLS, where they regulate the expression of certain genes under thecontrol of suitable promoters. In the plastids, the repressor binds toan artificial repressor binding site which has been introduced into theplastome and can thus repress the expression of the downstream gene (cf.WO 95/25787). In this manner it is possible, for example, to induce theexpression of a plastid-encoded DSBI enzyme when required, or to repressit until the point in time at which expression is desired.

Other promoters which are preferred are those which are induced bybiotic or abiotic stress such as, for example, the pathogen-induciblepromoter of the PRP1 gene (Ward et al., Plant Mol Biol 1993, 22:361-366), the heat-inducible tomato hsp70 promoter or hsp80 promoter(U.S. Pat. No. 5,187,267), the chill-inducible potato alpha-amylasepromoter (WO 96/12814) or the wounding-induced pinII promoter (EP-A 0375 091).

In an especially preferred embodiment, the nucleic acid which encodesthe DSBI enzyme is, above all, expressed under the control of aninducible promoter. A controlled expression capable of being governed isthus obtained, and any problems caused by expressing a DSBI enzymeconstitutively are avoided.

Advantageous control sequences for the expression cassettes or vectorsaccording to the invention comprise viral, bacteriophage or bacterialpromoters such as cos, tac, trp, tet, phoA, tat, lpp, lac, laciq, T7,T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL promoter. They are preferablyemployed in combination with the expression of the respective,corresponding RNA polymerase.

The expression in plastids can be effected using plastid promotersand/or transcription regulation elements. Examples which may bementioned, but not by way of limitation, are the RNA polymerase promoter(WO 97/06250) or the promoters described in WO 00/07431, U.S. Pat. No.5,877,402, WO 97/06250, WO 98/55595, WO 99/46394, WO 01/42441 and WO01/07590. The rpo B promoter element, the atpB promoter element, theclpP promoter element (see also WO 99/46394) or the 16S rDNA promoterelement should be mentioned. In this context, the promoter can also havea polycistronic “operon” assigned to it (EP-A 1 076 095; WO 00/20611).Systems in which a nonplant (for example viral) RNA polymerase isimported into the plastid using plastidic transit peptides andspecifically induces, in the plastid, the expression transgenicsequences which are under the control of the RNA polymerase recognitionsequences and have previously been inserted into the plastidic DNA havealso been described (WO 95/16783; U.S. Pat. No. 5,925,806; U.S. Pat. No.5,575,198).

In addition to the abovementioned promoters, the following can also bepreferably used:

-   a) the PrbcL promoter (SEQ ID NO: 44)-   b) the Prps16 promoter (SEQ ID NO: 50)-   c) the Prrn16 promoter (SEQ ID NO: 46)

In an especially preferred embodiment, NEP promoters are employed. Theseare promoters which are functional in plastids and are recognized by thenuclear-encoded plastidic RNA polymerases (NEP). The following arepreferred: Prrn-62; Pycf2-1577; PatpB-289; Prps2-152; Prps16-107;Pycf1-41; PatpI-207; PclpP-511; PclpP-173 and PaccD-129 (wo 97/06250;Hajdukiewicz P T J et al. (1997) EMBO J 16:4041-4048).

The following are especially preferred:

-   a) the PaccD-129 promoter of the tobacco accD gene (WO 97/06250; SEQ    ID NO: 47)-   b) the PclpP-53 promoter of the clpP gene as highly active NEP    promoter in chloroplasts (WO 97/06250; SEQ ID NO: 48)-   c) the Prrn-62 promoter of the rrn gene (SEQ ID NO: 49)-   d) the Prps16-107 promoter of the rps16 gene (SEQ ID NO: 45)-   e) the PatpB/E-290 promoter of the tobacco atpB/E gene (Kapoor S et    al. (1997) Plant J 11:327-337) (SEQ ID NO: 51)-   f) the PrpoB-345 promoter of the rpoB gene (Liere K & Maliga    P (1999) EMBO J 18: 249-257) (SEQ ID NO: 52)

In general, all those promoters which belong to class III (HajdukiewiczPTJ et al. (1997) EMBO J 16:4041-4048) and all fragments of the class IIpromoters which control the initiation of transcription by NEP can beutilized in this preferred embodiment. Such promoters or promotermoieties are not particularly highly conserved. ATAGAATAAA (SEQ ID NO:162) is given as consensus near the transcription initiation site of NEPpromoters (Hajdukiewicz PTJ et al (1997) EMBO J 16:4041-4048).

Normally, genes are surrounded by regulatory sequences which originatefrom the plastids of the organism to be transformed. Thus, sequenceduplications which can lead to instabilities owing to spontaneous,intrachromosomal homologous recombination events are generated (HeifetzP B (2000) Biochimie 82(6-7):655-666). To overcome this problem, it hasbeen proposed to utilize heterologous regulatory sequences or to exploitregulatory units which already exist endogenously in the plastidicgenome (WO 99/46394; WO 01/42441). A reduction of the homology bymutagenesis of the endogenous promoter sequence has also been described(WO 01/07590).

In principle, all natural promoters together with their regulatorysequences, such as those mentioned above, can be used for the methodaccording to the invention. Especially preferred promoters-are thosewhich have been isolated from prokaryotes. Very especially preferred arepromoters isolated from Synechocystis or E. coli. In addition, syntheticpromoters such as, for example, a synthetic promoter derived from the E.coli consensus sequence for σ70 promoters

5′-TTGACA N₁₆₋₁₉ TATAAT N₃ CAT-3′, (SEQ ID NO: 163)where N represents any nucleotide (that is to say A, G, C or T) canadditionally also be used advantageously. It is obvious to the skilledworker that individual or few base substitutions in the conservedregions stated are also possible without destroying the function of thepromoter. The variable design of these synthetic promoters by using avariety of sequential sequences makes it possible to generate amultiplicity of promoters which lack extensive homologies, whichincreases the stability of the expression cassettes in the plastome inparticular in the event that several promoters are required. Thefollowing, particularly preferred promoter sequences, which are derivedfrom the abovementioned consensus sequence, may be mentioned by way ofexample, but not by limitation:

(SEQ ID NO: 53) a) 5′-TTGACATTCACTCTTCAATTATCTATAATGATACA-3′ (SEQ ID NO:72) b) 5′-TTGACAATTTTCCTCTGAATTATATAATTAACAT-3′

It is obvious to the skilled worker that these synthetic promoters cancontrol the expression of any genes. For example, they can be utilizedfor driving the expression of a selection marker, also in order to beable to select under regenerative conditions for transplastomic plantswith the aid of said selection system. Examples of selection markers areenumerated further below. In addition, such synthetic promoters can belinked with any gene, for example with genes encoding antibodies,antigens or enzymes. Preferably, the expression cassettes consisting ofsuch promoters also comprise 5′-untranslated regions (or ribosomebinding sites) or 3′-noncoding regions which are detailed hereinbelow.

The invention furthermore relates to expression cassettes comprising anucleic acid sequence encoding a DSBI enzyme under the control of apromoter which is functional in plant plastids, for example one of theabove-described promoters. The expression cassette can comprise furtherelements such as, for example, transcription terminators and/orselection markers.

Genetic control sequences also comprise further promoters, promoterelements or minimal promoters which are capable of modifying theexpression-controlling properties. Genetic control sequences furthermorealso comprise the 5′-untranslated region (5′-UTR) or the noncoding 3′region (3′-UTR) of genes (Eibl C (1999) Plant J 19: 1-13). It has beendemonstrated that these can exert significant functions in regulatingthe gene expression in plastids of higher plants. In the nucleus, too,genetic control elements such as 5′-UTR, introns or 3′-UTR, can exert afunction in gene expression. Thus, for example, it has been demonstratedthat 5′-untranslated sequences can enhance the transient expression ofheterologous genes. They can furthermore promote tissue specificity(Rouster J et al., Plant J. 1998, 15: 435-440.).

5′-UTRs and 3′-UTRs which are preferably employed in plastids are:

-   a) 5′psbA (from tobacco) (SEQ ID NO: 54)-   b) 5′rbcL including 5′ portions from the coding region of the rbcL    gene (from tobacco) (SEQ ID NO: 55); the sequence described as SEQ    ID NO: 55 has been mutated in comparison with the native sequence in    order to introduce a PagI and an NcoI cleavage site.-   c) 5′rbcLs (SEQ ID NO: 56); the sequence described by SEQ ID NO: 56    has been mutated in comparison with the native sequence in order to    introduce a PagI cleavage site.-   d) 3′psbA-1 from Synechocystis (SEQ ID NO: 57)-   e) 3′psbA from tobacco (SEQ ID NO: 58)-   f) 3′rbcL from tobacco (SEQ ID NO: 59)

Genetic control sequences, especially for expression in plastids, alsocomprise in particular ribosome binding sequences for initiatingtranslation. They are usually present in the 5′-UTRs. This is especiallypreferred when suitable sequences are not provided by the nucleic acidsequence to be expressed or when such sequences are compatible with theexpression system. Especially preferred is the use of a syntheticribosome binding site (RBS) with the sequence 5′-GGAGG(N)₃₋₁₀ATG-3′,preferably 5′-GGAGG(N)₅ATG-3′ (SEQ ID NO: 60), particularly preferably5′-GGAGGATCTCATG-3′ (SEQ ID NO: 61).

The expression cassette can advantageously comprise one or more what areknown as enhancer sequences in operable linkage with the promoter; theseenhancer sequences make possible an enhanced transgenic expression ofthe nucleic acid sequence. Additional advantageous sequences, such asfurther regulatory elements or terminators, may also be inserted at the3′ end of the nucleic acid sequences to be expressed recombinantly. Oneor more copies of the nucleic acid sequences to be expressedrecombinantly may be present in the gene construct.

It is furthermore possible to insert, after the start codon, what isknown as a downstream box, which enhances expression in general(translation enhancer WO 00/07431; WO 01/21782).

Polyadenylation signals which are suitable as genetic control sequences,above all in the transformation of the nucleus, are plantpolyadenylation signals, preferably those which correspond essentiallyto T-DNA polyadenylation signals from Agrobacterium tumefaciens, inparticular of gene 3 of the T-DNA (octopine synthase) of the Ti plasmidpTiACHS (Gielen et al., EMBO J. 3 (1984), 835 et seq.) or functionalequivalents thereof. Examples of especially suitable terminatorsequences are the OCS (octopine synthase) terminator and the NOS(nopaline synthase) terminator.

The transformation vectors and insertion sequences according to theinvention may comprise further nucleic acid sequences. Such nucleic acidsequences can preferably constitute expression cassettes. The followingmay be mentioned by way of example of the DNA sequences to be expressedin the expression constructs, but not by limitation:

1. Selection Markers

“Selection markers” means all those nucleic acid or protein sequenceswhose expression (i.e. transcription and, if appropriate, translation)confers a phenotype to a cell, tissue or organism which differs fromthat of an untransformed cell, tissue or organism. Selection markerscomprises for example those nucleic acids or protein sequences whoseexpression confers an advantage (positiver selection marker) ordisadvantage (negative selection marker) on a cell, tissue or organismin comparison with cells which do not express this nucleic acid orprotein. For example, positive selection markers act by detoxifying asubstance which has an inhibitory effect on the cell (for exampleresistance to antibiotics/herbicides), or by forming a substance whichmakes possible improved regeneration or enhanced growth of the plantunder the selected conditions (for example nutritive markers,hormone-producing markers such as ipt; see hereinbelow). Another form ofpositive selection markers comprises mutated proteins or RNAs which areinsensitive to a selective agent (for example 16S rRNA mutants, whichare insensitive to spectinomycin). Negative selection markers act forexample by catalyzing the formation of a toxic substance in thetransformed cells (for example the codA gene). Moreover, selectionmarker can also comprise reporter proteins as long as they are suitablefor differentiating transformed from untransformed cells, tissues ororgans (for example by coloration or another detectable phenotype).

The following selection markers may be mentioned by way of example, butnot by limitation:

1.1 Positive Selection Markers:

The selectable marker introduced into the nucleus or the plastidstogether with the expression cassette confers resistance to a biocide(for example a herbicide such as phosphinothricin, glyphosate orbromoxynil), a metabolic inhibitor such as 2-deoxyglucose-6-phosphate(WO 98/45456) or an antibiotic such as, for example, tetracyclins,ampicillin, kanamycin, G 418, neomycin, bleomycin or hygromycin, to thesuccessfully transformed cells. The selection marker permits theselection of the transformed cells from untransformed cells (McCormicket al., Plant Cell Reports 5 (1986), 81-84; Dix PJ & Kavanagh TA (1995)Euphytica 85: 29-34).

Especially preferred selection markers are those which confer resistanceto herbicides. Selection markers which may be mentioned by way ofexample are:

-   -   DNA sequences which encode phosphinothricin acetyltransferases        (PAT), which acetylate the free amino group of the glutamine        synthase inhibitor phosphinothricin (PPT) and thus detoxify the        PPT (de Block et al. 1987, EMBO J. 6, 2513-2518) (also referred        to as Bialophos® resistance gene (bar)). The bar gene encoding a        phosphinothricin acetyltransferase (PAT) can be isolated for        example from Streptomyces hygroscopicus or S. viridochromogenes.        Such sequences are known to the skilled worker (from        Streptomyces hygroscopicus GenBank Acc. No.: X17220 and X05822,        from Streptomyces viridochromogenes GenBank Acc. No.: M 22827        and X65195; U.S. Pat. No. 5,489,520). Synthetic genes are        further described for example for expression in plastids. A        synthetic PAT gene is described in Becker et al. (1994) The        Plant J. 5:299-307. The genes confer resistance to the herbicide        bialaphos or glufosinate and are widely used markers in        transgenic plants (Vickers J E et al. (1996). Plant Mol Biol        Reporter 14:363-368; Thompson C J et al. (1987) EMBO J        6:2519-2523).    -   5-Enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase        genes), which confer resistance to glyphosate        (N-(phosphonomethyl)glycin). The nonselective herbicide        glyphosate has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS)        as molecular target. This enzyme has a key function in the        biosynthesis of aromatic amino acids in microbes and plants, but        not in mammals (Steinrucken H C et al. (1980) Biochem. Biophys.        Res. Commun. 94:1207-1212; Levin J G and Sprinson D B (1964) J.        Biol. Chem. 239: 1142-1150; Cole D J (1985) Mode of action of        glyphosate a literature analysis, p. 48-74. In: Grossbard E and        Atkinson D (eds.). The herbicide glyphosate. Buttersworths,        Boston.). Glyphosate-tolerant EPSPS variants are preferably used        as selection markers (Padgette S R et al. (1996). New weed        control opportunities: development of soybeans with a Roundup        Ready™ gene. In: Herbicide Resistant Crops (Duke, S.O., ed.),        pp. 53-84. CRC Press, Boca Raton, Fla.; Saroha M K and Malik V        S (1998) J Plant Biochemistry and Biotechnology 7:65-72). The        EPSPS gene of Agrobacterium sp. strain CP4 has a natural        tolerance to glyphosate which can be transferred to        corresponding transgenic plants. The CP4 EPSPS gene has been        cloned from Agrobacterium sp. strain CP4 (Padgette S R et        al. (1995) Crop Science 35(5):1451-1461). Sequences of        5-enolpyrvylshikimate-3-phosphate synthases which are        glyphosate-tolerant, such as, for example, those described in        U.S. Pat. No. 5,510,471; U.S. Pat. No. 5,776,760; U.S. Pat. No.        5,864,425; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,627;061;        U.S. Pat. No. 5,463,175; EP 0 218 571, are described in the        patents and also deposited in GenBank. Further sequences are        described under GenBank Accession X63374. The aroA gene        (M10947 S. typhimurium aroA locus        5-enolpyruvylshikimate-3-phosphate synthase (aroA protein) gene)        is furthermore preferred.    -   the gox gene (glyphosate oxidoreductase), which encodes the        Glyphosato® degrading enzyme. GOX (for example the glyphosate        oxidoreductase from Achromobacter sp.) catalyzes the cleavage of        a C—N bond in glyphosate, which is thus converted into        aminomethylphosphonic acid (AMPA) and glyoxylate. GOX can        thereby confer resistance to glyphosate (Padgette S R et        al. (1996) J Nutr. 1996 March; 126(3):702-16; Shah D et        al. (1986) Science 233: 478-481).    -   the deh gene (encoding a dehalogenase which inactives Dalapon®),        (GenBank Acc. No.: AX022822, AX022820 and WO 99/27116)    -   bxn genes, which encode bromoxynil-degrading nitrilase enzymes,        for example the Klebsiella ozanenae nitrilase. Sequences can be        found in GenBank for example under the Acc. No: E01313 (DNA        encoding bromoxynil specific nitrilase) and J03196 (K.        pneumoniae bromoxynil-specific nitrilase (bxn) gene, complete        cds).    -   Neomycin phosphotransferases confer resistance to antibiotics        (aminoglycosides) such as neomycin, G418, hygromycin,        paromomycin or kanamycin, by reducing their inhibitory action by        means of a phosphorylation reaction. Especially preferred is the        nptII gene. Sequences can be obtained from GenBank (AF080390        minitransposon mTn5-GNm; AF080389 minitransposon mTn5-Nm,        complete sequence). Moreover, the gene is already a component in        a large number of expression vectors and can be isolated from        them using methods with which the skilled worker is familiar        (such as, for example, polymerase chain reaction) (AF234316        pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314 pCAMBIA-2201). The        NPTII gene encodes an aminoglycoside 3′-O-phosphotransferase        from E. coli, Tn5 (GenBank Acc. No: U00004 position 1401-2300;        Beck et al. (1982) Gene 19-327-336). Moreover, the Acinetobacter        baumannii aphA-6 gene, which encodes an aminoglycoside        phospho-transferase, may also be utilized as selection marker        (Huang et al. (2002) Mol Genet Genomics 268:19-27)    -   the DOG^(R)1 gene. The gene DOG^(R)1 was isolated from the yeast        Saccharomyces cerevisiae (EP 0 807 836). It encodes a        2-deoxyglucose-6-phosphate phosphatase, which confers resistance        to 2-DOG (Randez-Gil et al. 1995, Yeast 11, 1233-1240; Sanz et        al. (1994) Yeast 10:1195-1202, sequence: GenBank Acc. No.:        NC001140 chromosome VIII, Saccharomyces cervisiae position        194799-194056).    -   Sulfonylurea- and imidazolinone-inactivating acetolactate        synthases, which confer resistance to imidazolinone/sulfonylurea        herbicides. Examples which may be mentioned of imidazolinone        herbicides are the active substances imazamethabenz-methyl,        imazamox, imazapyr, imazaquin and imazethapyr. Examples of        sulfonylurea herbicides which may be mentioned are        amidosulforon, azimsulfuron, chlorimuronethyl, chlorsulfuron,        cinosulfuron, imazosulfuron, oxasulfuron, prosulfuron,        rimsulfuron, sulfosulfuron. The skilled worker is familiar with        a large number of further active substances from the        abovementioned classes. Nucleic acid sequences such as, for        example, the sequence for the Arabidopsis thaliana Csr 1.2 Gen        (EC 4.1.3.18) which has been deposited under the GenBank Acc        No.: X51514, are suitable (Sathasivan K et al. (1990) Nucleic        Acids Res. 18(8):2188). Acetolactate synthases, which confer        resistance to imidazolinone herbicides, are furthermore        described under the GenBank Acc. Nos.:        -   a) AB049823 Oryza sativa ALS mRNA for acetolactate synthase,            complete cds, herbicide resistant biotype        -   b) AF094326 Bassia scoparia herbicide resistant acetolactate            synthase precursor (ALS) gene, complete cds        -   c) X07645 Tobacco acetolactate synthase gene, ALS SuRB (EC            4.1.3.18)        -   d) X07644 Tobacco acetolactate synthase gene, ALS SuRA (EC            4.1.3.18)        -   e) A19547 Synthetic nucleotide mutant acetolactate synthase        -   f) A19546 Synthetic nucleotide mutant acetolactate synthase        -   g) A19545 Synthetic nucleotide mutant acetolactate synthase        -   h) 105376 Sequence 5 from patent EP 0257993        -   i) 105373 Sequence 2 from patent EP 0257993        -   j) AL133315    -   Hygromycin phosphotransferases (X74325 P. pseudomallei gene for        hygromycin phosphotransferase) which confer resistance to the        antibiotic hygromycin. The gene is a component of a large number        of expression vectors and can be isolated from them using        methods with which the skilled worker is familiar (such as, for        example, polymerase chain reaction) (AF294981 pINDEX4; AF234301        pCAMBIA-1380; AF234300 pCAMBIA-1304; AF234299 pCAMBIA-1303;        AF234298 pCAMBIA-1302; AF354046 pCAMBIA-1305.; AF354045        pCAMBIA-1305.1)    -   genes for resistance to        -   a) chloramphenicol (chloramphenicol acetyltransferase),        -   b) tetracyclin, various resistance genes have been            described, for example X65876 S. ordonez genes class D tetA            and tetR for tetracyclin resistance and repressor proteins            X51366 Bacillus cereus plasmid pBC16 tetracyclin resistance            gene. Moreover, the gene is already a component of a large            number of expression vectors and can be isolated therefrom            using methods known to the skilled worker (such as, for            example polymerase chain reaction)        -   c) Streptomycin; various resistance genes have been            described, for example with the GenBank Acc. No.:AJ278607            Corynebacterium acetoacidophilum ant gene for streptomycin            adenylyltransferase.        -   d) Zeocin; the corresponding resistance gene is a component            of a large number of cloning vectors (for example L36849            cloning vector pZEO) and can be isolated from these using            methods known to the skilled worker (such as, for example,            polymerase chain reaction).        -   e) Ampicillin (β-lactamase gene; Datta N, Richmond            M H. (1966) Biochem J. 98(1):204-9; Heffron F et            al. (1975) J. Bacteriol 122: 250-256; the Amp gene was first            cloned for generating the E. coli vector pBR322; Bolivar F            et al. (1977) Gene 2:95-114). The sequence is a component of            a large number of cloning vectors and can be isolated from            them using methods known to the skilled worker (such as, for            example, polymerase chain reaction).    -   Genes such as the isopentenyl transferase from Agrobacterium        tumefaciens (strain:PO22) (Genbank Acc. No.: AB025109). The ipt        gene is a key enzyme of cytokinin biosynthesis. Its        overexpression facilitates the regeneration of plants (for        example selection on cytokinin-free medium). The method for        utilizing the ipt gene has been described (Ebinuma H et        al. (2000) Proc Natl Acad Sci USA 94:2117-2121; Ebinuma, H et        al. (2000) Selection of marker-free transgenic plants using the        oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable        markers, In Molecular Biology of Woody Plants. Kluwer Academic        Publishers).

Various other positive selection markers which confer a growth-relatedadvantage to the transformed plants over the nontransformed plants, andmethods for their use, have been described, inter alia, in EP-A 0 601092. Examples which may be mentioned are β-glucuronidase (in conjunctionwith, for example, cytokinin glucuronide), mannose-6-phosphate isomerase(in conjunction with mannose), UDP-galactose 4-epimerase (in conjunctionwith, for example, galactose), with mannose-6-phosphate isomerase inconjunction with mannose being especially preferred.

Preferred for a selection marker which is functional in plastids are, inparticular, those which confer resistance to spectinomycin,streptomycin, kanamycin, lincomycin, gentamycin, hygromycin,methotrexate, bleomycin, phleomycin, blasticidin, sulfonamide,phosphinothricin, chlorsulfuron, bromoxynil, glyphosate, 2,4-D,atrazine, 4-methyltryptophan, nitrate, S-aminoethyl-L-cysteine,lysine/threonine, aminoethyl-cysteine or betaine aldehyde. Especiallypreferred are the genes aadA, nptII, BADH, FLARE-S (a fusion of aadA andGFP, described by Khan M S & Maliga P, 1999 Nature Biotech 17: 910-915).

As selection marker which is functional in plastids, it is mainly theaadA gene which has been described (Svab Z and Maliga P (1993) Proc NatlAcad Sci USA 90:913-917). Also described are modified 16S rDNA, thenptII gene (kanamycin resistance) and the bar gene (phosphinothricinresistance). Owing to the preference given to the selection marker aadA,the latter is preferably recycled, i.e. deleted from the genome, orplastome, following its use (Fischer N et al. (1996) Mol Gen Genet251:373-380; Corneille S et al. (2001) Plant J 27:171-178), so that aadacan be reused as selection marker in further transformations of analready transplastomic plant. The betaine-aldehyde dehydrogenase (BADH)from spinach has been described as a further possible selection marker(Daniell H et al. (2001) Trends Plant Science 6:237-239; Daniell H etal. (2001) Curr Genet 39:109-116; WO 01/64023; WO 01/64024; WO01/64850). Lethal agents such as, for example, glyphosate, can also beutilized in connection with correspondingly detoxifying or resistantenzymes (WO 01/81605).

Binding type markers may also be utilized. To utilize the DBSrecognition sequence of the homing endonuclease I-CpaI in the gene ofthe 23S rRNA, which sequence is preferred as insertion site, at leastthe 3′ end of the insertion sequence (preferably an artificial intron)is surrounded by homologous sequences of the target region. Thus,sequences of the 23S rDNA are incorporated into the transformationvector. Point mutations can be introduced at one position (position 2073or 2074 of the tobacco 23S rRNA, sequence: AAAGACCCTATGAAG becomessequence: GGAGACCCTATGAAG), which point mutations confer resistance tolincomycin to the ribosomes derived from a 23S rDNA which has beenmutated thus (Cseplö A et al. (1988) Mol Gen Genet 214:295-299). Furtherpoint mutations comprise those in the tobacco 16S rRNA which conferreistance to spectinomycin (mutation underlined):

a) 5′-GGAAGGTGAGGATGC-3′ (A in native sequence)

Other mutations confer resistance to streptomycin:

b) 5′-GAATGAAACTA-3′ (C in native sequence)1.2 Negative Selection Markers

Negative selection markers make possible for example the selection oforganisms with successfully deleted sequences which comprise the markergene (Koprek T et al. (1999) The Plant Journal 19(6):719-726). Forexample, sequences which encode selection markers or DSBI enzymes can bedeleted from the genome/plastome after successful application of themethod according to the invention.

When carrying out a negative selection, for example a compound whichotherwise has no disadvantageous effect on the plant is converted into acompound which is disadvantageous, for example owing to the negativeselection marker introduced into the plant.

Genes which have a disadvantageous effect per se are furthermore alsosuitable, such as, for example, TK thymidine kinase (TK) and diphtheriatoxin A fragment (DT-A), the coda gene product encoding a cytosinedeaminase (Gleave A P et al. (1999) Plant Mol Biol 40(2):223-35; PereraR J et al. (1993) Plant Mol Biol 23(4): 793-799; Stougaard J (1993)Plant J 3:755-761), the cytochrome P450 gene (Koprek et al. (1999) PlantJ. 16:719-726), genes encoding a haloalkane dehalogenase (Naested H(1999) Plant J. 18:571-576), the iaaH gene (Sundaresan V et al. (1995)Genes & Development 9:1797-1810) or the tms2 gene (Fedoroff N V & SmithD L (1993) Plant J 3:273-289).

The concentrations-of the antibiotics, herbicides, biocides or toxinswhich are used in each case to carry out the selection must be adaptedto the respective test-conditions or organisms. Examples which may bementioned in context with plants are: kanamycin (Km) 50 to 100 mg/l,hygromycin B 40 mg/l, phosphino-thricin (Ppt) 6 to 20 mg/l,spectinomycin (Spec) 15 to 500 mg/l.

Furthermore, it is possible to express functional analogs of theabovementioned nucleic acids encoding selection markers. Functionalanalogs means, in the present context, all those sequences which haveessentially the same function, i.e. which are capable of selectingtransformed organisms. In this context, it is quite feasible that thefunctional analog differs with regard to other characteristics. Forexample, it can have a higher or lower activity, or else have furtherfunctionalities.

Functional analogs means furthermore sequences which encode fusionproteins consisting of one of the preferred selection markers andanother protein, for example a further preferred selection marker, oneof the reporter proteins mentioned hereinbelow or a PLS. By way ofexample, a fusion of the GFP (green fluorescent protein) and the aadagene may be mentioned (Sidorov V A et al. (1999) Plant J 19:209-216).

2. Reporter Genes

Reporter genes encode readily quantifiable proteins which, via theircolor or enzyme activity, allow an assessment of the transformationefficiency, the site or time of expression or the identification oftransgenic plants. Very especially preferred in this context are genesencoding reporter proteins (see also Schenborn E, Groskreutz D. MolBiotechnol. 1999; 13(1):29-44) such as

-   -   “green fluorescent protein” (GFP). (Chui W L et al., Curr Biol        1996, 6:325-330; Leffel S M et al., Biotechniques. 23(5):912-8,        1997; Sheen et al. (1995) Plant Journal 8(5):777-784; Haseloff        et al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et        al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et        al. (1997) Plant Cell Rep 16:267-271; WO 97/41228).    -   Chloramphenicol transferase,    -   Luciferase (Millar et al., Plant Mol Biol Rep 1992 10:324-414;        Ow et al. (1986) Science, 234:856-859); allows detection via        bioluminescence.    -   β-Galactosidase, encodes an enzyme for which a variety of        chromogenic substrates are available.    -   β-Glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6,        3901-3907) or the uida gene, which encodes an enzyme for a        variety of chromogenic substrates.    -   R-Locus gene product: protein which regulates the production of        anthocyanin pigments (red coloration) in plant tissue and thus        makes possible the direct analysis of the promoter activity        without addition of further auxiliary substances or chromogenic        substrates (Dellaporta et al., In: Chromosome Structure and        Function: Impact of New Concepts, 18th Stadler Genetics        Symposium, 11:263-282, 1988).    -   β-Lactamase (Sutcliffe (1978) Proc Natl Acad Sci USA        75:3737-3741), enzyme for a variety of chromogenic substrates        (for example PADAC, a chromogenic cephalosporin).    -   xy1E gene product (Zukowsky et al. (1983) Proc Natl Acad Sci USA        80:1101-1105), catechol dioxygenase capable of converting        chromogenic catechols.    -   Alpha-amylase (Ikuta et al. (1990) Bio/technol. 8:241-242).    -   Tyrosinase (Katz et al. (1983) J Gen Microbiol 129:2703-2714),        enzyme which oxidizes tyrosine to DOPA and dopaquinone, which        subsequently form melanin, which can be detected readily.    -   Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun        126(3):1259-1268), can be used in the calcium-sensitive        bioluminescence detection.

The selection marker, or the reporter gene, is preferably encoded on thetransformation construct, especially preferably on the insertionsequence. However, it can also be encoded on an independenttransformation construct which is introduced into the nucleus or theplastids of a plant cell in the form of a cotransformation together withthe transformation construct of interest.

The transformation vectors and insertion sequences according to theinvention may comprise further functional elements. The concept offurther functional elements is to be understood in the broad sense.Preferably, it is understood as meaning all those elements whichinfluence the generation, multiplication, function, use or value of theinsertion sequences, transformation constructs or transformation vectorsused within the scope of the present invention. The following may bementioned by way of example of further functional elements, but not bylimitation:

-   -   i. Replication origins (ORI) which make possible an        amplification of the expression cassettes or vectors according        to the invention in, for example, E. coli or else in plastids.        Examples of E. coli ORIs which may be mentioned are the pBR322        ori, the P15A ori (Sambrook et al.: Molecular Cloning. A        Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press,        Cold Spring Harbor, N.Y., 1989) or the colE1 ORI, for example        from pBLUESCRIPT. Plastidic ORIs are described in U.S. Pat. No.        5,693,507, U.S. Pat. No. 5,932,479 or WO 99/10513.    -   ii. Multiple cloning regions (MCRs) permit and facilitate the        insertion of one or more nucleic acid sequences.    -   iii. Sequences which make possible the homologous recombination        or insertion into the genome or plastome of a host organism.    -   iv. Elements, for example border sequences, which make possible        the Agrobacterium-mediated transfer into plant cells for the        transfer and integration into the plant genome, such as, for        example, the right or left border of the T-DNA or the vir        region.

An insertion sequence or an expression construct for a DSBI enzyme canbe inserted advantageously using vectors into which these constructs, orcassettes, are inserted. Vectors can be plasmids, cosmids, phages,viruses, retroviruses or else agrobacteria, by way of example.

In an advantageous embodiment, the expression cassette is inserted bymeans of plasmid vectors. Preferred vectors are those which makepossible a stable integration of the expression cassette into the hostgenome or plastome.

The generation of a transformed organism or a transformed cell requiresintroducing the DNA in question into the host cell in question, or intothe plastids thereof. A multiplicity of methods is available for thisprocedure, which is referred to as transformation (see also Keown et al.(1990) Methods in Enzymology 185:527-537). Thus, for example, the DNAcan be introduced directly by microinjection, electroporation or bybombardment with DNA-coated microparticles. Also, the cell can bepermeabilized chemically, for example with polyethylene glycol, so thatthe DNA can penetrate the cell by diffusion. Transformation can also beeffected by fusion with other DNA-comprising units such as minicells,cells, lysosomes or liposomes. Others which must additionally bementioned are transfection using calcium phosphate, DEAE dextran orcationic lipids, transduction, infection, the incubation of dry embryosin DNA-comprising solution, sonication, and the transformation of intactcells or tissue by microinjection or macroinjection into tissue orembryos, or tissue electroporation, or the vacuum infiltration of seeds.The skilled worker is familiar with such methods. In the case ofinjection or electroporation of DNA into plant cells, the plasmid usedneed not meet any particular requirements. Simple plasmids such as thosefrom the pUC series can be used. If intact plants are to be regeneratedfrom the transformed cells, the presence of an additional, selectablemarker gene on the plasmid is useful. Methods for the regeneration ofplants from plant tissues or plant cells have also been described.

There are several options for introducing DNA into the plastids. Theonly decisive aspect for the present invention is that the DNA isintroduced into the plastids. However, the present invention is notlimited to a specific method. Any method which permits the introductionof the DNA to be transformed into the plastids of a higher plant issuitable. The stable transformation of plastids is a method with whichthe skilled worker is familiar; it has been described for higher plants(inter alia by Svab Z and Maliga P (1993) Proc Natl Acad Sci USA90(3):913-917). For example, the methods are based on transformation bymeans of the particle gun and insertion into the plastidic genome byhomologous recombination under selection pressure. Further methods aredescribed in U.S. Pat. No. 5,877,402. In EP-A 0 251 654, the DNA isintroduced by Agrobacterium tumefaciens (see De Block M et al. (1985)EMBO J 4:1367-1372; Venkateswarlu K and Nazar R N (1991) Bio/Technology9:1103-1105). It has furthermore been demonstrated that DNA can beintroduced into isolated chloroplasts by means of electroporation, thusbringing about transient expression (To KY et al. (1996) Plant J10:737-743). Transformation by means of a direct DNA transfer intoplastids of protoplasts, for example using PEG (polyethylene glycol) ispreferred (Koop H U et al. (1996) Planta 199:193-201; Kofer W et al.(1998) In Vitro Cell Dev Biol Plant 34:303-309; Dix P J and Kavanagh T A(1995) Euphytica. 85:29-34; EP-A 0 223 247). Most preferred arebiolistic transformation methods. Here, the DNA to be transformed isapplied to, for example gold or tungsten particles. These particles aresubsequently accelerated towards the explant to be transformed (Dix P Jand Kavanagh T A (1995) Euphytica. 85:29-34; EP-A 0 223 247).Thereafter, transplastomic plants are regenerated under selectionpressure on suitable medium in the manner with which the skilled workeris familiar. Such methods have been described (for example U.S. Pat. No.5,451,513; U.S. Pat. No. 5,877,402; Svab Z et al. (1990) Proc Natl AcadSci USA 87:8526-8530; Svab Z and Maliga P (1993) Proc Natl Acad Sci USA90:913-917). Moreover, the DNA can be introduced into the plastids bymeans of microinjection. A specific microinjection method has beendescribed recently (Knoblauch M et al. (1999) Nature Biotech 17:906-909;van Bel AJE et al. (2001) Curr Opin Biotechnol 12:144-149). This methodis particularly preferred for the present invention. It is also possibleto introduce, by means of protoplast fusion, the plastids from onespecies into a different species, to transform them in the latter andsubsequently to return them to the original species by protoplast fusion(WO 01/70939).

Besides these “direct” transformation techniques, transformation canalso be carried out by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes (Horsch R B (1986) Proc NatlAcad Sci USA 83(8):2571-2575; Fraley et al. (1983) Proc Natl Acad SciUSA 80:4803-4807; Bevans et al. (1983) Nature 304:184-187). Theexpression cassette for, for example, the DSBI enzyme is preferablyintergrated into specific plasmids, either into a shuttle, orintermediate, vector or into a binary vector. Binary vectors arepreferably used. Binary vectors are capable of replication both in E.coli and in Agrobacterium and be transformed directly into Agrobacterium(Holsters et al. (1978) Mol Gen Genet 163:181-187). Various binaryvectors are known; some of them are commercially available such as, forexample, pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al. (1984)Nucl Acids Res 12:8711). The selection marker gene permits the selectionof transformed agrobacteria and is, for example, the nptII gene, whichconfers resistance to kanamycin. The binary plasmid can be transferredinto the agrobacterial strain for example by electroporation or othertransformation methods (Mozo & Hooykaas 1991, Plant Mol. Biol. 16,917-918). The plant explants are generally cocultured with theagrobacterial strain for two to three days. The agrobacterium which, inthis case, acts as the host organism, should already comprise a plasmidwith the vir region. Many Agrobacterium tumefaciens strains are capableof transferring genetic material, such as, for example, the strainsEHA101[pEHA101] (Hood E E et al. (1996) J Bacteriol 168(3):1291-1301),EHA105[pEHA105] (Hood et al. (1993) Transgenic Research 2:208-218),LBA4404[pAL4404] (Hoekema et al. (1983) Nature 303:179-181),C58C1[pMP90] (Koncz and Schell (1986) Mol Gen Genet 204:383-396) andC58C1[pGV2260] (Deblaere et al. (1985) Nucl Acids Res 13: 4777-4788).

To transfer the DNA to the plant cell, plant explants are coculturedwith Agrobacterium tumefaciens or Agrobacterium rhizogenes. Startingfrom infected plant material (for example leaf, root or stem portions,but also protoplasts or suspensions of plant cells), intact plants canbe regenerated using a suitable medium which may comprise, for example,antiobiotics or biocides for selecting transformed cells. Acotransformed selection marker permits the selection of transformedcells from untransformed cells (McCormick et al. (1986) Plant CellReports 5:81-84). The plants obtained can be bred, selfed and hybridizedin the customary manner. Two or more generations should be grown toensure that the genomic integration is stable and hereditary. Theabovementioned methods are described in, for example, Jenes B et al.(1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, edited by Kung S D and Wu R, AcademicPress, pp. 128-143 and in Potrykus (1991) Ann Rev Plant Physiol PlantMolec Biol 42:205-225).

The Agrobacterium-mediated transformation is best suited todicotyledonous plant cells, whereas the direct transformation techniquesare suitable for any cell type.

The Agrobacterium-mediated transformation is especially preferablyemployed for the transformation of the nucleus, while the directtransformation techniques are especially preferably employed for thetransformation of plastids.

As soon as a predominanly homotransplastomic plant cell has beengenerated by the method according to the invention, an intact plant canbe obtained using methods with which the skilled worker is familiar. Thestarting material for this purpose is, for example, callus cultures. Thedevelopment of shoot and root can be induced in the known manner in thisas yet undifferentiated biomass. The shoots obtained can be planted outand used for breeding.

Deletion Methods

In the above-described methods according to the invention, it isadvantageous, at various levels, to remove certain sequences which havepreviously been introduced (for example sequences for selection-markersand/or DSBI enzymes) from the plastome or genome of the plant or cell.Thus, it is advantageous, but not necessarily required, to remove theselection marker which has been introduced for example during theinsertion of a normatural DSB recognition sequence, from the masterplant since the same selection marker can then be utilized in asubsequent transformation (for example with the insertion sequence).Deletion is particularly advantageous since the selection marker is nolonger necessarily required after the selection phase, and thereforesuperfluous. Moreover, deletion increases the consumer acceptance and isdesirable for registration purposes. Moreover, the protein synthesisapparatus of the plastid is not unnecessarily burdened by the synthesisof the marker protein, which has potentially advantageous effects on thecharacteristics of the plant in question.

The skilled worker is familiar with a variety of methods for thedirected deletion of sequences. One which should be mentioned by way ofexample, but not by limition, is the excision by means of recombinases.Various sequence-specific recombination systems have been described,such as the Cre/lox system of bacteriophage P1 (Dale E C and Ow D W(1991) Proc Natl Acad Sci USA 88:10558-10562; Russell S H et al. (1992)Mol Gen Genet 234: 49-59; Osborne B I et al. (1995) Plant J. 7,687-701), the yeast FLP/FRT system (Kilby N J et al. (1995) Plant J8:637-652; Lyznik L A et al. (1996) Nucleic Acids Res 24:3784-3789), theGin recombinase of the phage Mu, the Pin recombinase from E. coli or theR/RS system of the plasmid pSR1 (Onouchi H et al. (1995) Mol Gen Genet247:653-660; Sugita K et al. (2000) Plant J 22:461-469). These methodscan be utilized not only for deleting DNA sequences from the nucleargenome, but also from the plastome (Corneille et al. (2001) Plant J 27:171-178; Hajdukieicz et al. (2001) Plant J 27:161-170). Furtherrecombinases which can be employed are, for example, PhiC31 (Kuhstoss &Rao (1991) J Mol Biol 222:897-908), TP901 (Christiansen et al. (1996) JBacteriol 178:5164-5173), xisF from Anabaena (Ramaswamy et al. (1997)Mol Microbiol 23:1241-1249), integrase from phage PhiLC3 (Lillehaug etal. (1997) Gene 188:129-136) or the recombinase encoded by the sre geneof the R4 phage (Matsuura et al. (1996) J Bacteriol 178:3374-3376).

In a preferred embodiment, however, the deletion is effected byintrachromosomal recombination owing to suitably introduced sequenceduplications. The efficiency of the latter can be enhanced by thedirected introduction of double-strand breaks near the sequenceduplications (cf. FIG. 8). To this end, the sequence to be deleted isflanked bilaterally by homology sequences H1 and H2 which havesufficient length and homology to undergo recombination with oneanother. Recombination is induced by the induction of at least onesequence-specific double-strand break of a further DSB recognitionsequence located near one of the two homology sequences (but preferablydifferent from the first one). This DSB recognition sequence ispreferably localized between the two homology sequences. To induce thedouble-strand break, it is preferred to express or introduce a secondDSBI enzyme which differs from the first one. This method is especiallypreferably utilized for deleting selection markers from the plastome.

The invention furthermore relates to the transplastomic, predominantlyhomoplastomic, plants generated using the method according to theinvention, and to parts of these plants, such as leaves, roots, seeds,fruits, tubers, pollen or cell cultures, callus and the like which arederived from such plants.

The invention furthermore relates to the plants employed in the methodaccording to the invention which comprise an expression cassetteaccording to the invention for a DSBI enzyme or a fusion protein of PLSand DSBI enzyme. In this context, the expression cassette for the fusionprotein of PLS and DSBI enzyme is—especially preferably—stablyintegrated in the nuclear DNA under the control of a promoter which isfunctional in the plant nucleus. The expression cassette encoding a DSBIenzyme under the control of a promoter which is active in plant plastidsis preferably stably integrated into the plastome. Comprised arefurthermore parts of same such as leaves, roots, seeds, tubers, fruits,pollen or cell cultures, callus and the like which are derived from theabovementioned plants.

Genetically modified plants according to the invention which can beconsumed by humans and animals can also be used as foods or feeds, forexample directly or following processing known per se.

The invention furthermore relates to the use of the above-describedtransplastomic, predominantly homoplastomic, plants according to theinvention and of the cells, cell cultures, parts—such as, for example,the roots, leaves and the like in the case of transgenic plantorganisms—and transgenic propagation material such as seeds or fruitswhich are derived from them for the production of foods or feeds,pharmaceuticals or fine chemicals.

Fine chemicals refers to enzymes such as, for example, the industrialenzymes mentioned hereinbelow, vitamins such as, for example,tocopherols and tocotrienols (for example vitamin E) and vitamin B2,amino acids such as, for example, methionine, lysine or glutamate,carbohydrates such as, for example, starch, amylose, amylopectin orsucrose, fatty acids such as, for example, saturated, unsaturated andpolyunsaturated fatty acids, natural and synthetic flavorings, aromachemicals such as, for example linalool, menthol, borneone (camphor),pinene, limonene or geraniol, and colorants such as, for examle,retinoids (for example vitamin A), flavonoids (for example quercetin,rutin, tangeretin, nobiletin) or carotenoids (for example β-carotene,lycopene, astaxanthin). The production of tocopherols and tocotrienolesand of carotenoids is especially preferred. Growing the transformed hostorganisms, and isolation from the host organisms or the growth medium,are carried out using methods with which the skilled worker is familiar.The production of pharmaceuticals such as, for example, antibodies orvaccines has been described (Hood E E, Jilka J M. (1999) Curr OpinBiotechnol. 10(4):382-386; Ma J K and Vine N D (1999) Curr Top MicrobiolImmunol. 236:275-92).

The method according to the invention is particularly suitable forproducing industrial enzymes within what is known as “phytofarming”.Examples of industrial enzymes which may be mentioned, but not by way oflimitation, are lipases, esterases, proteases, nitrilases, acylases,epoxyhydrolases, amidases, phosphatases, xylanases, alcoholdehydrogenases, amylases, glucosidases, galactosidases, pullulanases,endocellulases, glucanases, cellulases, nucleases, chitin deacetylases,monoaminooxidases, lysozymes and laccases.

Embodiments which are especially preferred for the purposes of theinvention are described in greater detail hereinbelow within theexplanations of the figures.

Sequences

-   1. SEQ ID NO: 1    -   pCB42-94 Basic vector for plastid transformation.-   2. SEQ ID NO:2    -   Nucleic acid sequence inserted into the multiple cloning site of        pCB42-94 (SEQ ID NO: 1). Resulting vector: pCB199-3.-   3. SEQ ID NO:3    -   Nucleic acid sequence inserted into the multiple cloning site of        pCB42-94 (SEQ ID NO: 1). Resulting vector: pCB401-20-   4. SEQ ID NO:4    -   Expession cassette from pCB289-13 for the expression of the        I-PpoI homing endonuclease in plastids.-   5. SEQ ID NO:5    -   Amino acid sequence of the I-PpoI homing endonuclease encoded by        the expression cassette from pCB289-13.-   6. SEQ ID NO:6    -   XhoI/BglII fragment employed for generating the vector        pCB304-25.-   7. SEQ ID NO:7    -   Nucleic acid sequence inserted into the multiple cloning site of        pGEMTeasy. Resulting vector: pCB220-17-   8. SEQ ID NO:8    -   Nucleic acid sequence inserted into the multiple cloning site of        pBluescript. Resulting vector: pCB270-1-   9. SEQ ID NO:9    -   Sequence from vector pCB315-1: LacZ gene with inserted intron        for detecting splicing.

10. SEQ ID NO: 10

-   -   Ll.LtrB intron from vector pCB345-34.

-   11. SEQ ID NO: 11    -   Synthetic sequence of the homing endonuclease I-PpoI (ORF: 16 to        507)

-   12. SEQ ID NO: 12    -   Protein sequence of the homing endonuclease I-PpoI

-   13. SEQ ID NO: 13    -   Nucleic acid sequence of the homing endonuclease I-CpaI from        Chlamydomonas pallidostigmatica (modification of the published        sequence at position 69. An NcoI cleavage site was introdued at        ATG (ORF: 4 to 462)

-   14. SEQ ID NO: 14    -   Protein sequence of the homing endonuclease I-CpaI

-   15. SEQ ID NO: 15    -   Sequence comprising the CpLSU2 intron

-   16. SEQ ID NO: 16: Oligonucleotide primer p19

5′-TAAGGCCCTCGGTAGCAACGG-3′

-   17. SEQ ID NO: 17: Oligonucleotide primer p20

5′-GGGGTACCAAATCCAACTAG-3′

-   18. SEQ ID NO: 18: Oligonucleotide primer p21:

5′-GGAGCTCGCTCCCCCGCCGTCGTTC-3′

-   19. SEQ ID NO: 19: Oligonucleotide primer p22

5′-GATGCATGATGACTTGACGGCATCCTC-3′

-   20. SEQ ID NO: 20: Oligonucleotide primer p190

5′-GTCGACAGATCTTTAA-3′

-   21. SEQ ID NO: 21: Oligonucleotide primer p191

5′-AGATCTGTCGACTTAA-3′

-   22. SEQ ID NO: 22: Oligonucleotide primer p199

5′-GATCTCCAGTTAACTGGGGTAC-3′

-   23. SEQ ID NO: 23: Oligonucleotide primer p200

5′-CCCAGTTAACTGGA-3′

-   24. SEQ ID NO: 24: Oligonucleotide primer p218

5′-TTAAGCCAGTTAACTGGGCGGAGCT-3′

-   25. SEQ ID NO: 25: Oligonucleotide primer p219

5′-CCGCCCAGTTAACTGGC-3′

-   26. SEQ ID NO: 26: Oligonucleotide primer p276

5′-TCGAGAAGATCAGCCTGTTATCCCTAGAGTAACT-3′

-   27. SEQ ID NO: 27: Oligonucleotide primer p277

5′-CTAGAGTTACTCTAGGGATAACAGGCTGATCTTC-3′

-   28. SEQ ID NO: 28: Oligonucleotide primer p91

5′-AGAAGACGATCCTAAGG-3′

-   29. SEQ ID NO: 29: Oligonucleotide primer p92

5′-TGAAGACTTGACAAGGAATTTCGC-3′

-   30. SEQ ID NO: 30: Oligonucleotide primer p102

5′-AGAAGACGATCCTAAATAGCAATATTTACCTTTGGGACCAAAAGTTA TCAGGCATG-3

-   31. SEQ ID NO: 31: Oligonucleotide primer p103

5′TGAAGACTTGACAAGGAATTTCGCTACCTTCGAGTACTCCAAAACTAA TC-3′

-   32. SEQ ID NO: 32: Oligonucleotide primer p207

5′-GAGAAGACATTCCTAACACATCCATAACGTGCG-3′

-   33. SEQ ID NO: 33: Oligonucleotide primer p208

5′-TGAAGACTTGACATTTGATATGGTGAAGTAGG-3′

-   34. SEQ JD NO: 34    -   Nucleic acid sequence encoding the transit peptide of the small        subunit (SSU) of ribulose bisphosphate carboxylase (Rubisco ssu)        from pea-   35. SEQ ID NO: 35    -   Transit peptide of the small subunit (SSU) of ribulose        bisphosphate carboxylase (Rubisco ssu) from pea-   36. SEQ ID NO: 36    -   Transit peptide of the tobacco plastidic transketolase.-   37. SEQ ID NO: 37    -   Nucleic acid sequence encoding the transit peptide of the        tobacco plastidic transketolase (reading frame 1; pTP09)-   38. SEQ ID NO: 38    -   Nucleic acid sequence encoding the transit peptide of the        tobacco plastidic transketolase (reading frame 2; pTP10)-   39. SEQ ID NO: 39    -   Nucleic acid sequence encoding the transit peptide of the        tobacco plastidic transketolase (reading frame 3; pTP11)-   40. SEQ ID NO: 40    -   Transit peptide of the plastidic isopentenyl-pyrophosphate        isomerase-2 (IPP-2) from Arabidopsis thaliana.-   41. SEQ ID NO: 41    -   Nucleic acid sequence encoding the transit peptide of the        plastidic isopentenyl-pyrophosphate isomerase-2 (IPP-2) from        Arabidopsis thaliana (reading frame 1; IPP-9)-   42. SEQ ID NO: 42    -   Nucleic acid sequence encoding the transit peptide of the        plastidic isopentenyl-pyrophosphate isomerase-2 (IPP-2)        Arabidopsis thaliana (reading frame 2; IPP-10)-   43. SEQ ID NO: 43    -   Nucleic acid sequence encoding the transit peptide of the        plastidic isopentenyl-pyrophosphate isomerase-2 (IPP-2) from        Arabidopsis thaliana (reading frame 3; IPP-11)-   44. SEQ ID NO: 44    -   Nucleic acid sequence encoding the tobacco PrbcL promoter.-   45. SEQ ID NO: 45    -   Nucleic acid sequence encoding the tobacco Prps16-107 promoter.-   46. SEQ ID NO: 46    -   Nucleic acid sequence encoding the tobacco Prrn16 promoter.-   47. SEQ ID NO: 47    -   Nucleic acid sequence encoding the tobacco PaccD-129 promoter.-   48. SEQ ID NO: 48    -   Nucleic acid sequence encoding the tobacco PclpP-53 promoter.-   49. SEQ ID NO: 49    -   Nucleic acid sequence encoding the tobacco Prrn-62 promoter.-   50. SEQ ID NO: 50    -   Nucleic acid sequence encoding the tobacco Prps16 promoter.-   51. SEQ ID NO: 51    -   Nucleic acid sequence encoding the tobacco PatpB/E-290 promoter.-   52. SEQ ID NO: 52    -   Nucleic acid sequence encoding the tobacco PrpoB-345 promoter.-   53. SEQ ID NO: 53    -   Nucleic acid sequence encoding a promoter derived from the        consensus sequence of the E. coli σ70 promoters.-   54. SEQ ID NO: 54    -   Nucleic acid sequence encoding the 5′-untranslated region of the        tobacco psbA gene (5′psbA)-   55. SEQ ID NO: 55    -   Nucleic acid sequence encoding the 5′-untranslated region        including 5′ portions from the coding region of the tobacco rbcL        gene (5′rbcL).-   56. SEQ ID NO: 56    -   Nucleic acid sequence encoding the 5′-untranslated region of the        tobacco rbcLs gene.-   57. SEQ ID NO: 57    -   Nucleic acid sequence encoding the 3′-untranslated region of the        Synechocystis psbA-1 gene (3′psbA-1)-   58. SEQ ID NO: 58    -   Nucleic acid sequence encoding the 3′-untranslated region of the        tobacco psbA gene (3′psbA)-   59. SEQ ID NO: 59    -   Nucleic acid sequence encoding the 3′-untranslated region of the        tobacco rbcL gene (3′rbcL)-   60. SEQ ID NO: 60    -   Nucleic acid sequence encoding synthetic ribosome binding sites        (RBS)-   61. SEQ ID NO: 61    -   Nucleic acid sequence encoding synthetic ribosome binding sites        (RBS)-   62. SEQ ID NO: 62    -   Complete insert of the vector pCB304-25-   63. SEQ ID NO: 63    -   BglII/MunI fragment of the vector pCB320-192.-   64. SEQ ID NO: 64: Oligonucleotide primer p93

5′-AAAGATCTCCTCACAAAGGGGGTCG-3′

-   65. SEQ ID NO: 65: Oligonucleotide primer p97

5′-TCGAAGACTTAGGACCGTTATAG-3′

-   66. SEQ ID NO: 66: Oligonucleotide primer p98

5′-AGGAAGACCTTGTCGGGTAAGTTCCG-3′

-   67. SEQ ID NO: 67: Oligonucleotide primer p95:

5′-CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG-3′

-   68. SEQ ID NO: 68: Nucleic acid sequence encoding fusion proteins    from the native I-Ppo-I nuclease and the IPP plastid localization    sequence (ORF for I-PpoI: 181-672; IPP transit peptide: 1-180;    native sequence from 1-172).-   69. SEQ ID NO: 69: Fusion proteins of the native I-Ppo-I nuclease    and the IPP-plastid localization sequence.-   70. SEQ ID NO: 70: Nucleic acid sequence encoding long version of    the I-Ppoi homing endonuclease.-   71. SEQ ID NO: 71: Amino acid sequence encoding long version of the    I-PpoI homing endonuclease.-   72. SEQ ID NO: 72: Nucleic acid sequence encoding a promoter    sequence derived from the consensus sequence of the σ70 promoters    from E. coli.-   73. SEQ ID NO: 73: Nucleic acid sequence encoding the artificial    intron TetIVS2a.-   74. SEQ ID NO: 74: Insert of vector pCB459-1-   75. SEQ ID NO: 75: Insert of vector pCB478-3-   76. SEQ ID NO: 76: Insert of vector pCB492-25-   77. SEQ ID NO: 77: Oligonucleotide primer p396

5′-TAGTAAATGACAATTTTCCTCTGAATTATATAATTAACATGGCGACT GTTTACCAAAAAC-3

-   78. SEQ ID NO: 78: Oligonucleotide primer p95    5′-CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG-3′-   79. SEQ ID NO: 79: Nucleic acid sequence encoding PCR product    Prom-TetIVS2a-Cpa-   80. SEQ ID NO: 80: Insert of vector pCB435-45-   81. SEQ ID NO: 81: Nucleic acid sequence encoding probe for Southern    blot analysis (directed against portions of the 16SrDNA).-   82. SEQ ID NO: 82: Nucleic acid sequence encoding probe for Southern    blot analysis (directed against portions of the 23SrDNA).-   83. SEQ ID NO: 83: Insert of vector pCB456-2-   84. SEQ ID NO: 84: Insert of vector pCB528-2 from KpnI to SacI

Figures

Within the method according to the invention, particularly theembodiments detailed in the figures hereinbelow are especiallypreferred. The following abbreviations are generally used in thefigures:

-   A, A′ Pair of homologous sequences A and A′-   A/A′ Result of a homologous recombination between A and A′ and/or a    substitution of A by A′ caused by repair synthesis.-   B, B′ Pair of homologous sequences B and B′-   B/B′ Result of a homologous recombination between B and B′ and/or a    substitution of B by B′ caused by repair synthesis.-   H1, H2: Pair of homologous sequences H1 and H2-   H1/2: Sequence as the result of the homologous recombination of H1    and H2-   DS Functional DSB recognition sequence-   nDS nonfunctional half of a DSB recognition sequence-   E: DSBI enzyme-   P: Promoter-   I: Further nucleic acid sequence (gene of interest)-   S, S′ Positive selection markers-   NS Negative selection marker-   IS Intron sequences. The intron in total is marked as a box. The box    comprises all elements required for a functional intron.

As already described above, A/A′ and B/B′ are the result of a homologousrecombination and/or a substitution brought about by repair synthesis.The resulting sequence, in turn, can be the starting sequence forfurther homologous recombinations or repair syntheses. For the sake ofsimplicity, this sequence (A/A′ and B/B′) is again referred to as A andB, respectively, in the steps which follow.

1. FIG. 1: Introduction of a DSB Recognition Sequence into the Plastomeby Means of Double Cross-Over

In an especially preferred embodiment 1, a DSBR construct is firstintroduced into plastids of a higher plant. In this embodiment, the DSBRconstruct is preferably equipped with homologous target regions and withan expressible selection marker (promoter—5′UTR—selection marker—3′UTR)and, in this embodiment, preferably comprises a recognition region for aDSBI enzyme which preferably has no natural recognition sequence in theplastidic genome of the (untransformed) plant in question. The DSBRconstruct can optionally already encode further genes of interest.Predominantly homoplastomic master plants are generated (FIG. 1).

2. FIG. 2A-E: Introduction of an Insert Sequence with an ExpressionCassette for a DSBI Enzyme and, if Appropriate, Selection Markers andFurther Genes of Interest

Explants of the master plants generated in embodiment 1 are utilized fora further transformation with a transformation construct according tothe invention. Preferably, the transformation construct according to theinvention has regions which are homologous to the sequences surroundingthe insertion site of the DSBR construct, which regions are preferablylocated on both sides (FIGS. 2A, 2B) or on one side (FIGS. 2C, 2D) ofthe insertion sequence. Insertion now takes place via homologousrecombination (for example cross-over) or via repair synthesis.

The sequences to be inserted are especially preferably—following thehomology sequences inwardly—flanked by portions of the DSB recognitionsequence (nDS) which correspond to the portions originating as theconsequence of cleavage with the DSBI enzyme (FIG. 2B). The insertionsequence thus comprises sequences which correspond in detail to the endswhich are the result of a cleavage in the plastome and thus ensure aparticularly efficient incorporation.

If the transformation construct, or the insertion sequence, has no suchhomologous regions, the insertion sequence is preferably provided, atthese ends, with overhangs which are also generated by the DSBI enzymeafter cleavage of the master plant plastome (FIG. 2E).

If only one homology sequence is present, this sequence borders, in anespecially preferred embodiment, an nDS sequence (see hereinabove asdescribed for FIG. 2B), while the other side of the insertion sequenceis provided with overhangs which correspond to those generated by theDSBI enzyme in the plastome of the master plant (FIG. 2D).

The insertion sequence optionally codes for a further expressibleselection marker which differs functionally from that of the DSBRconstruct, if appropriate one or more expressible genes of interest andthe expressible DSBI enzyme, which cleaves the recognition sequenceintroduced by the DSBR construct at the insert site in the plastidicgenome of the master plant. The insertion sequence of the transformationconstruct is inserted, in this context, in such a way at a position thatsaid recognition region is no longer functional after the insertion.

3. FIG. 3: Introduction of a DSB Recognition Sequence into the Plastomeby Means of Double Cross-Over in a Transcriptionally Active Region

In a further, especially preferred embodiment 2, a DSBR construct isinitially introduced into plastids of a higher plant. In thisembodiment, the DSBR construct is preferably equipped with homologoustarget regions and with an expressible selection marker, an endogenouspromoter of the plastome being utilized, and it preferably additionallycomprises a recognition sequence for a DSBI enzyme which preferably hasno natural recognition sequence in the plastidic genome of the(nontransformed) plant in question. The DSBR construct may alreadyencode genes of interest. Predominantly homoplastomic master plants aregenerated (FIG. 3).

4. FIG. 4A-E: Introduction of an Isertion Sequence with a CassetteEncoding a DSBI Enzyme and, if Appropriate, a Selection Marker andFurther Genes of Interest

Explants of the master plants generated in embodiment 2 are utilized fora further transformation with a transformation construct according tothe invention. Preferably, the transformation construct according to theinvention has regions which are homologous to the sequences surroundingthe insertion site of the DSBR construct, which regions are preferablylocated on both sides (FIGS. 4A, 4B) or on one side (FIGS. 4C, 4D) ofthe insertion sequence. Insertion now takes place via homologousrecombination (for example cross-over) or via repair synthesis. Thesequences to be inserted are especially preferably—following thehomology sequences inwardly—flanked by portions of the DSB recognitionsequence (nDS) which correspond to the portions originating as theconsequence of cleavage with the DSBI enzyme (FIG. 4B). The insertionsequence thus comprises sequences which correspond in detail to the endswhich are the result of a cleavage in the plastome and thus ensure aparticularly efficient incorporation.

If the transformation construct, or the insertion sequence, has no suchhomologous regions, the insertion sequence is preferably provided, atthese ends, with overhangs which are also generated by the DSBI enzymeafter cleavage of the master plant plastome (FIG. 4E).

If only one homology sequence is present, this sequence borders, in anespecially preferred embodiment, an nDS sequence (see hereinabove asdescribed for FIG. 4B), while the other side of the insertion sequenceis provided with overhangs which correspond to those generated by theDSBI enzyme in the plastome of the master plant (FIG. 4D).

The insertion sequence optionally codes for a further expressibleselection marker which differs functionally from that of the DSBRconstruct, if appropriate one or more expressible genes of interest andthe expressible DSBI enzyme, which cleaves the recognition sequenceintroduced by the DSBR construct at the insert site in the plastidicgenome of the master plant. The insertion sequence of the transformationconstruct is inserted, in this context, in such a way at a position thatsaid recognition region is no longer functional after the insertion.

5. FIG. 5A-E: Introduction of an Insertion Sequence with a CassetteEncoding a DSBI Enzyme and, if Appropriate, Selection Markers andFurther Genes of Interest Utilizing Natural, Endogenous DSB RecognitionSequences

In a further, very especially preferred embodiment 3, a transformationconstruct according to the invention comprises an expressible DSBIenzyme which has an endogenous, natural recognition sequence in theplastome of the plant in question.

Explants of these natural master plants are utilized for atransformation with a transformation construct according to theinvention. Preferably, the transformation construct according to theinvention has regions which are homologous to the sequences surroundingthe insertion site of the DSBR construct, which regions are preferablylocated on both sides (FIGS. 5A, 5B) or on one side (FIGS. 5C, 5D) ofthe insertion sequence. Insertion now takes place via homologousrecombination (for example cross-over) or via repair synthesis.

The sequences to be inserted are especially preferably—following thehomology sequences inwardly—flanked by portions of the DSB recognitionsequence (nDS) which correspond to the portions originating as theconsequence of cleavage with the DSBI enzyme (FIG. 5B). The insertionsequence thus comprises sequences which correspond in detail to the endswhich are the result of a cleavage in the plastome and thus ensure aparticularly efficient incorporation.

If the transformation construct, or the insertion sequence, has no suchhomologous regions, the insertion sequence is preferably provided, atthese ends, with overhangs which are also generated by the DSBI enzymeafter cleavage of the master plant plastome (FIG. 5E).

If only one homology sequence is present, this sequence borders, in anespecially preferred embodiment, an nDS sequence (see hereinabove asdescribed for FIG. 5B), while the other side of the insertion sequenceis provided with overhangs which correspond to those generated by theDSBI enzyme in the plastome of the master plant (FIG. 5D).

The insertion sequence of the tranformation construct is, in thiscontext, preferably inserted at a position in such a way that saidrecognition region is no longer functional after the insertion. Theinsertion sequence preferably encodes an expressible selection marker(S′), one or more genes of interest, and the expressible DSBI enzyme.The selection marker is optional.

6. FIG. 6A-E: Introduction of an Insertion Sequence with a CassetteEncoding Genes of Interest and, if Appropriate, Selection Markers, andIntroduction of a DSBI Enzyme in Trans

In further preferred embodiments 4, the DSBI enzyme is not encoded bythe transformation construct, but either expressed in trans (in plastidsor as PLS fusion protein in the nucleus) or transfected into theplastids in the form of RNA or protein. The DSBI enzyme recognizeseither an artificially introduced DSB recognition sequence (FIGS. 6A,6B) or a natural DSB recognition sequence (FIGS. 6C, 6D). Thisembodiment is especially preferred when the transformation constructcomprises no promoter elements, and expression of the coded genes isonly realized after insertion into the plastome, using plastidic,endogenous promoters.

As was the case in the embodiments which have already been describedabove, the transformation construct preferably has regions bilaterally(FIGS. 6A, 6B) or unilaterally (not shown) of the insertion sequencewhich are homologous to the sequences surrounding the insertion site ofthe DSBR construct. Insertion now takes place via homologousrecombination (for example cross-over) or repair synthesis.

The sequences to be inserted are especially preferably—following thehomology sequences inwardly—flanked by portions of the DSB recognitionsequence (nDS) which correspond to the portions originating as theconsequence of cleavage with the DSBI enzyme (FIGS. 6B, 6D). Theinsertion sequence thus comprises sequences which correspond in detailto the ends which are the result of a cleavage in the plastome and thusensure a particularly efficient incorporation.

If the transformation construct, or the insertion sequence, has no suchhomologous regions, the insertion sequence is preferably provided, atthese ends, with overhangs which are also generated by the DSBI enzymeafter cleavage of the master plant plastome (FIG. 6E). Thetransformation construct can additionally comprise a sequence encoding aDSBI enzyme. However, expression only takes place after successfulinsertion into the plastome, so that it is desirable that a first amountof functional RNA or protein of a DSBI enzyme is provided.

If only one homology sequence is present, it borders, in an especiallypreferred embodiment, an nDS sequence (see above as described for FIGS.6B, 6D), while the other side of the insertion sequence is provided withoverhangs which correspond to those generated by the DSBI enzyme in theplastome of the master plant (not shown).

In this context, the insertion sequence of the transformation constructis preferably inserted at a position in such a way that said recognitionregion is no longer functional after the insertion.

7. FIG. 7A-E: Introduction of an Insertion Sequence Comprising an IntronSequence with a Cassette Encoding Genes of Interest and, if Appropriate,Selection Markers or DSBI Enzymes

In a further, very especially preferred embodiment 5, the gene ofinterest (and optionally a selection marker S′ and/or the DSBI enzyme)is/are encoded within an intron which is functional at the insertionsite selected, i.e. which can splice out of the transcript formedtherein.

Preferably, the transformation construct according to the invention hasregions which are homologous to the sequences surrounding the insertionsite of the DSBR construct, which regions are preferably located on bothsides (FIGS. 7A, 7B) or one one side (not shown) of the insertionsequence. Insertion now takes place via homologous recombination (forexample cross-over) or via repair synthesis.

If the transformation construct or the insertion sequence has no suchhomologous regions, the insertion sequence is preferably provided, atthese ends, with overhangs which are also generated by the DSBI enzymeafter cleaving the plastome of the master plant (not shown).

Expression can be controlled by means of a promoter which is present onthe transformation construct (FIG. 7B) or an endogenous, plastidicpromoter (FIG. 7A). In the first case, the DSBI enzyme is preferablypresent on the transformation construct (FIG. 7B), while, in the lattercase, it is either expressed (at least in parallel) in trans (inplastids or as PLS fusion protein in the nucleus) or transfected intothe plastids in the form of RNA or protein (FIG. 7A).

In this context, the insertion sequence of the transformation constructis preferably inserted at a position in such a way that said recognitionregion is no longer functional after the insertion. The transformationconstruct can additionally optionally comprise a sequence encoding aDSBI enzyme. However, expression only takes place after successfulinsertion into the plastome, so that it is desirable that a first amountof functional RNA or protein of a DSBI enzyme is provided.

8. FIG. 8: Deletion of Sequences by Means of Intramolecular HomologousRecombination Induced by Sequence-Specific Double-Strand Breaks

In all of the above-described embodiments, sequences—for example thoseencoding selection markers or DSBI enzymes—are preferably flanked byhomology sequences H1 and H2 with sufficient length and homology toundergo recombination with one another. The recombination is induced bythe induction of at least one double-strand break in the DSB recognitionsequence located between the two homology sequences. To induce thedouble-strand break, it is preferred to transiently express or introducea DSBI enzyme (FIG. 8).

The skilled worker realizes that the sequence of the genes expressed inan operon is exchangeable and can thus vary in the above-describedembodiments. Also, when using only one homology sequence for insertingthe insertion sequence, this homology sequence may be localized at the5′ side (as shown in the figures) or the 3′ side of the double-strandbreak. In principle, the DSBI enzyme can be expressed on thetransformation construct and/or separately (in the nucleus or theplastids) and introduced differently into—plastids, for example bytransfection with RNA or protein.

9. FIG. 9: Southern Analysis of Predominantly Homotransplastomic Plants

Wild-type and predominantly homotransplastomic master plants wereanalyzed with regard to the modification (introduction of a DSBrecognition sequence; cf. Example 4). Owing to the modification, a 1750bp band was detected (lanes 2, 3 and 4 corresponding to linesCB199NTH-4, -6 and -8), while a 3100 bp band was detected in theunmodified wild-type plant (lane 1).

10. FIG. 10: Modification of the IGS of the Tetrahymena LSU Intron.

Capital letters indicate the sequence of the intron, while lower-caseletters represent the sequence of the surrounding exons. The flankingexon sequences, the 5′ and 3′ portion of the intron or intronderivative, and the sequence comprising the IGS are shown. Bars betweenthe bases indicate possible base pairings which can be formed forinitiating the splicing procedure.

-   A: The abovementioned sequence segments of the naturally occurring    Tetrahymena LSU intron in its natural exon environment are shown    (SEO ID NO: 160).-   B: The abovementioned sequence segments of the Tetrahymena LSU    intron derivative generated within the scope of the present    invention (TetIVS2a) in the above-defined exon environment, as is    found in the CpLSU5 intron within the DSB recognition sequence of    the DSBI enzyme I-CpaI are shown (SEO ID NO: 161). Letters in bold    represent the mutations carried out in comparison with the natural    sequence.    11. FIG. 11:-   A: Southern analysis with BamHI-cut total DNA from the tobacco lines    CB255+435NTH-16b, -16c, -19 and -20. A region of the 16Sr DNA was    used as probe. The bands representing plastome copies which    correspond to the wild type (WT; approx. 3.2 kb band detected) and    those which bear the transgene (TG; approx. 2.3 kb band detected)    are identified by arrows.-   B: Schematic representation of transplastomic tobacco plants which    have originated by the insertion of the insertion sequence from    pCB435-45; and the bands to be expected in a corresponding Southern    analysis (cf. A).-   C: Southern analysis with HindIII-cut total DNA from the tobacco    lines CB255+435NTH-16b, -16c, -19 and -20. A region of the 23Sr DNA    was used as probe. The bands representing plastome copies which    correspond to the wild type (WT; approx. 1.1 kb band detected) and    those which bear the transgene (TG; approx. 1.5 kb band detected)    are identified by arrows.-   D: Schematic representation of transplastomic tobacco plants which    have originated by the insertion of the insertion sequence from    pCB255-1; and the bands to be expected in a corresponding Southern    analysis (cf. C).    12. FIG. 12:-   A: wild-type and predominantly homotransplastomic master plants were    analyzed in a Southern analysis with regard to the modification    (introduction of one of the I-PpoI DSB recognition sequence; cf.    Example 14.2). Owing to the modification, an approximately 1.7 kb    band was detected in the DNA which had been treated here with EcoRI    (TG; lanes 1 and 4 corresponding to lines CB456NTH-1 and -15), while    an approximately 3.1 kb band was detected in the unmodified    wild-type plant (WT; lane 6). (wt—unmodified wild-type plant; wild    type—shows the expected fragment size in unmodified wild-type    plants; transgenic—shows the expected fragment size in plants    CB456NTH)-   B: Schematic representation of the EcoRI fragment which was to be    expected in A by hybridization with the probe in a modified plant    CB456NTH. (trnV—gene encoding a tRNA-Val; rrn16—gene encoding the    16SrRNA; aada—gene encoding a selection marker; 3′psbA    (Synec)—noncoding region upstream of the Synechocystis psbA-1 gene,    here incorporated into the expression cassette for the selection    marker aada; Psynth.—synthetic promoter derived from the consensus    sequence for E. coli σ70 promoters; DSB-R: DSB recognition    sequence).

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

EXAMPLES

General Methods:

Oligonucleotides can be synthesized chemically for example in the knownmanner, using the phosphoamidite method (Voet, Voet, 2nd edition, WileyPress New York, pages 896-897). The cloning steps carried out within thescope of the present invention such as, for example, restrictioncleavages, agarose gel electrophoresis, purification of DNA fragments,transfer of nucleic acids to nitrocellulose and nylon membranes, linkageof DNA fragments, transformation of E. coli cells, bacterial cultures,propagation of phages, and the sequence analysis of recombinant DNA, arecarried out as described by Sambrook et al. (1989) Cold Spring HarborLaboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules aresequenced with an ALF-Express laser fluorescence DNA sequencer(Pharmacia, Uppsala, Sweden) following the method of Sanger (Sanger etal. (1977) Proc Natl Acad Sci USA 74:5463-5467).

Example 1 Generating a Basic Vector for the Transformation of Plastids

Initially, the selected target regions were cloned from the plastome oftobacco cv. SR1 by means of PCR. The left-hand target region wasamplified using the primers p19 and p20.

p19: 5′-TAAGGCCCTCGGTAGCAACGG-3′ (SEQ ID NO: 16) p20:5′-GGGGTACCAAATCCAACTAG-3′ (SEQ ID NO: 17)

The primers p21 and p22 were used for amplifying the right-hand targetregion, the last-mentioned primer additionally introducing aspectinomycin resistance into the amplfied part of the 16S rDNA, inaddition to the SR1 resistance (binding-type marker).

(SEQ ID NO: 18) p21: 5′-GGAGCTCGCTCCCCCGCCGTCGTTC-3′ (SEQ ID NO: 19)p22: 5′-GATGCATGATGACTTGACGGCATCCTC-3′

The two amplified regions were cloned into pBluescript and pzeroBlunt,respectively, and sequenced. The left-hand and right-hand target regionswere subsequently cloned into the backbone of the pUC19 vector. Thecleavage sites EcoO109I and PvuII of the vector were used for thispurpose. A multiple cloning site from pBluescript (from KpnI to SacI)was cloned between the left-hand and right-hand target region. Thismultiple cloning site resides between the two plastid-encoded genes trnvand rrnl6 in the basic vector for the transformation of plastids.

This basic vector for the transformation of plastids was named pCB42-94(SEQ ID NO: 1). The vector-comprises the following sequence elements:

-   a) Position complementary to bp 55-1405: right-hand target    region-with-the partial gene of the 16S rRNA (complementary to bp 56    to 1322). The latter comprises mutations for the streptomycin    resistance (SR1, position bp 346) and spectinomycin resistance    (SPC1, position bp 68).-   b) Position complementary to bp 2374 to 1510: left-hand target    region comprising ORF131 (bp 1729 to 2124) and trnv gene inter alia    (complementary to bp 1613 to 1542).-   c) Position bp 1404 to 1511: multiple cloning site-   d) Position bp 2629 to 3417: ampicillin resistance in the vector    backbone

Example 2 Generation of a Vector (pCB199-3) for Introducing aNon-Naturally Occurring Recognition Region for the Homing EndonucleasesI-PpoI into the Plastome of Tobacco

Various elements were cloned one after the other into the multiplecloning site of the basic vector pCB42-94 (SEQ ID NO: 1) for thetransformation of plastids:

-   a) frt recognition region (mutated, contains no XbaI cleavage site;    complementary to 1307-1354)-   b) expression cassette for expressing the marker gene aada,    consisting of:    -   i) promoter of the gene for 16S rRNA (complementary to        1191-1281)    -   ii) 5′-untranslated region of the tobacco rbcL gene        (complementary to 1167-1184) including mutated 5′ portions of        the rbcL gene (duplication of 6 AS of the rbcL gene, partly        mutated, as the consequence of the cloning strategy so that a        fusion encoding a total of 12 amino acids (complementary to        1131-1166) with the subsequent element, the aada gene, was        formed)    -   iii) aada gene (complementary to 336-1130)    -   iv) the 3′ region of the psbA gene (complementary to 232-323)-   c) core recognition region for the homing endonuclease I-PpoI    (complementary to. 176-190).

This vector, which is referred to as pCB199-3, comprises theabovementioned elements within the nucleic acid sequence with the SEQ IDNO: 2 instead of the multiple cloning site in the basic vector for thetransformation of plastids. The sequence which replaces the complete MCSfrom KpnI to SacI is shown. However, there is no longer a KpnI cleavagesite in the sequence shown, owing the cloning strategy.

Example 3 Generation of a Further Vector (pCB401-20) for Introducing aNon-Naturally Occurring Recognition region for the Homing EndonucleaseI-PpoI into the Plastome of Tobacco

In contrast to the vector pCB199-3 described in Example 2, the vectordescribed herein comprises no promoter and 3′UTR linked directly to theselection marker aada. Rather, the expression of the aada gene iscontrolled starting from the promoter of the trnV gene which islocalized in the plastidic genome or in the left-hand target regionupstream of the aadA gene. The purpose of generating this vector was toavoid sequence duplication by exploiting regulatory regions from thetobacco plastidic genome. To this end, various elements were cloned oneafter the other into the multiple cloning site of the basic vectorpCB42-94 for the transformation of plastids:

-   a) ribosome binding site (complementary to bp 1033 to 1050)-   b) aada gene (complementary to bp 238 to 1032)-   c) core recognition region for the homing endonuclease I-PpoI    (complementary to bp 176 to 190)

The resulting vector also confers spectinomycin resistance in E. coli.This vector, which is referred to as pCB401-20, comprises theabovementioned elements within the nucleic acid sequence with the SEQ IDNO: 3 instead of the multiple cloning site in the basic vector pCB42-94(SEQ ID NO: 1) for the transformation of plastids. Again, all of thesequence which replaces the MCS (from SacI to KpnI) is shown.

Example 4 Generation of Predominantly Homoplastomic Tobacco MasterPlants Comprising a Non-Natural DSB Recognition Sequence

The plasmid pCB199-3 was introduced into the plastids of tobacco(Nicotiana tabacum cv. Petit Havana) as described hereinbelow. Theregenerated plants-were named CB199NTH. Independent lines were providedwith different last numbers (for example CB199NTH-4).

The vector pCB401-20 is introduced analogously into the plastids oftobacco. Accordingly, the resulting plants are named CB401NTH.

First, leaf disks of diameter 2.0 to 2.5 cm were punched out ofin-vitro-cultured plants, using a sterile cork borer, and placedupside-down on a Petri dish with bombardment medium [MS salts(Sigma-Aldrich): 4.3 g/l; sucrose: 30.0 g/l, Phyto-agar (Duchefa,P1003): 0.6% (w/v); pH 5.8; after autoclaving, 1.0 mg/l thiamine(Duchefa, T0614) and 0.1 g/l myo-inositol (Duchefa, I0609,) were added].The underside of the leaf, which faced away from the agar, wassubsequently bombarded using the particle gun. To this end, the plasmidDNA to be transformed (isolated from E. coli using Nucleobond AX100Macherey & Nagel) was first applied to gold particles 0.6 μm in side bythe following protocol (“coating”). First, 30 mg of powdered gold(BioRad) were taken up in ethanol. 60 μl of the gold suspension weretransferred into a fresh Eppendorf tube and the gold particles weresedimented by centrifugation (for 10 seconds). The gold particles werewashed twice in each case 200 μl of sterile water and, after a furthercentrifugation step, taken up in 55 μl of water. The following wereadded rapidly, with continuous mixing (vortexing):

-   -   5 μl plasmid DNA (1 μg/μl)    -   50 μl 2.5 M CaCl₂    -   20 μl 0.1 M spermidine

The suspension was subsequently vortexed for a further 3 minutes andsubsequently centrifuged briefly. The gold/DNA complexes which hadsedimented were washed once or twice in each case 200 μl of ethanol and,after a further centrifugation step, finally taken up in 63 μl ofethanol. 3.5 μl (corresponding to 100 μl of gold) of this suspensionwere applied to a macrocarrier for each bombardment.

The particle gun (BioRad, PDS1000He) was prepared in accordance with themanufacturer's instructions, and the leaf explants were bombarded withthe gold/DNA complexes from a distance of 10 cm. The followingparameters were used: vacuum: 27 inch Hg, pressure 1100 psi. After thebombardment, the explants were incubated for 2 days incontrolled-evironment cabinets (24° C., 16 h light, 8 h darkness) andsubsequently divided into segments approximately 0.5 cm² in size, usinga surgical blade. These segments were then transferred to regenerationmedium [bombardment medium supplemented with 1 mg/l 6-benzylaminopurine(BAP, Duchefa, B0904) and 0.1 mg/l naphthylacetic acid (NAA, Duchefa,N0903)] supplemented with 500 mg/l spectinomycin (Duchefa, S0188) andincubated for 10 to 14 days under the abovementioned conditions in acontrolled-environment cabinet. After this period of time had elapsed,the leaf segments were transferred to fresh regeneration mediumsupplemented with 500 mg/l spectinomycin. This procedure was repeateduntil green shoots formed on the explants. The shoots were removed usinga surgical blade and grown on growth medium (like bombardment medium,but with 10 g/l sucrose instead of 30 g/l sucrose) supplemented with 500mg/l spectinomycin.

To obtain as predominantly homoplastomic plants as possible, it isoptionally possible to excise explants from the regenerated plantsthemselves and to place them on regeneration medium with 1000 mg/lspectinomycin. Regenerating shoots are transferred into glass containerswith growth medium supplemented with 500 to 1000 mg/l spectinomycin.After the plants have rooted, they are transferred into the greenhouse,where they are grown in soil until the seeds have matured.

When the transformation was carried out with the plasmid pCB199-3, 8plants with resistance to spectinomycin were obtained. PCR and Southernanalyses proved that three of these lines (lines CB199NTH-4, -6 and -8)have indeed incorporated the aada gene into the plastidic genome.

To perform a Southern analysis of the transplastomic plants, total DNAfrom leaves was isolated from transformed and untransformed plants withthe aid of the GenElute Plant Genomic DNA Kit (Sigma). The DNA was takenup in 200 μl of eluate. 86 μl portions of this were treated with in eachcase 10 μl of 10× restriction puffer and 40 U of restrictionendonuclease and incubated for 4 to 8 hours at the temperaturerecommended for the restriction enzyme. The DNA was subsequentlyprecipitated with ethanol in the manner known to the skilled worker andthe precipitate was subsequently taken up in 20 μl of water. The sampleswere subsequently separated on agarose gel by methods known to theskilled worker, and the DNA was denatured in the gel and transferred toa nylon membrane by means of a capillary blot.

A suitable probe for the radioactive hybridization was generated withthe aid of the HighPrime (Roche) system. First, the membrane wasprehybridized for 1 hour at 65° C. with Hyb buffer (1% (w/v))bovine-serum-albumin; 7% (w/v) SDS; 1 mM EDTA; 0.5 M sodium phosphatebuffer, pH 7.2). The heat-denatured probe was subsequently added andleft to hybridize overnight at 65° C. The blots were subsequently washedas follows: one rinse with 2×SSPE/0.1% SDS; washing for 15 minutes at65° C. with 2×SSPE/0.1% SDS; washing for 15 minutes at 65° C. with1×SSPE/0.1% SDS; if appropriate, the last step was repeated again(20×SSPE is 3 M NaCl; 0.2 M NaH₂PO₄; 0.5 M EDTA; pH 7.4).

The hybridization was subsequently analyzed with the aid of aphosphoimager (Molecular Imager FX, BioRad).

For example, PstI-cut total DNA from different plants which had beenregenerated after transformation with pCB199-3 were hybridized with theaadA gene as radiolabeled probe (793 bp PstI/NcoI fragment frompCB199-3). Here, it was found that the lines CB199NTH-4, -6 and -8 hadindeed incorporated the aadA gene into the DNA. Moreover, EcoRI- andXhoI-cut total DNA from CB199NTH-4, -6 and -8 was hybridized with aradiolabeled probe (1082 bp Bsp120I/SacI fragment from pCB199-3), whichhybridizes with part of the 16S rDNA. While, as expected, anapproximately 3100 bp band was detected in the wild type (untransformedplant), mostly a band at 1750 bp was detected in the transplastomiclines, as the result of the insertion of the insertion sequence frompCB199-3 into the plastome (FIG. 9). The resulting plants can beconsidered as being predominantly homotransplastomic.

Example 5 Generation of Transformation Vectors which can be used forTransforming the Plastids of Master Plants CB199NTH by Means of theArtificial Homing Process

5.1 Cloning of the Homing Endonuclease I-PpoI

The homing endonuclease I-PpoI was generated from 26 syntheticoliogonucleotides by means of PCR, following a modification of themethod of Stemmer WPC et al. (1995) Gene 164: 49-53 (SEQ ID NO: 11). Thebasic sequence was derived from the published sequence (Accession No.M38131 nucleotides 86 to 577). Here, a few mutations were introduced toremove restriction endonuklease recognition sites from the gene;however, these mutations did not involve an altered amino acid sequence.The following elements were subsequently combined one after the other ina pBluescript KS (Stratagene) vector backbone in order to generate anI-PpoI expression cassette. The sequence is flanked by the cleavagesites KpnI and SacI.

-   a) Posit-ion 21 to 111: Prrn promoter-   b) Position 118 to 135: 5′-untranslated region of the rbcL gene    followed by 18 bp encoding 6 amino acids of the rbcL protein (bp    136-152)-   c) Position 154 to 645: Nucleic acid sequence encoding I-PpoI.-   d) Position 688-779: 3′-untranslated region of the psbA gene.

The resulting plasmid was named pCB289-13. Despite the expression of theenzyme I-PpoI, which was expected to take place in E. coli, no adverseeffects on the growth were observed. The sequence described by SEQ IDNO: 4 resulted from the KpnI cleavage site to the SacI cleavage site(vector backbone remains that of pBluescript KS).

5.2 Generation of a Transformation Vector for Artificial Homing withHomologous Regions Flanking the Insertion Sequence Bilaterally

I) Without I-PpoI in the Insertion Sequence

Regions around the I-PpoI recognition region from pCB199-3 were excisedusing PstI and SacI and ligated into the PstI and SacI cleavage sites ofpBluescript. Thereafter, cleavage sites which ere not required wereremoved from this vector by linearizing it with PstI and Bsp120I and,following treatment with Klenow fragment, recircularizing the vector.With the aid of commercially available enzyme I-PpoI (PROMEGA GmbH,Mannheim, Germany), the corresponding recognition region was cleaved inthe resulting vector, and further cleavage sites were inserted thereinby means of the synthetic oligos p190 and p191.

Oligo p190: 5′-GTCGACAGATCTTTAA-3′ (SEQ ID NO: 20) Oligo p191:5′-AGATCTGTCGACTTAA-3′ (SEQ ID NO: 21)

An expression cassette consisting of the following elements wasintroduced in the form of.a BglII/XhoI fragment (SEQ ID NO: 6) into thecleavage sites SalI and BglII, which had thus been introduced:

-   a) Prps16 promoter (complementary to 1033-1139)-   b) 5′rbcL (complementary to 1007-1024) with 18 bp encoding the 6 AAs    (complementary to 989-1006)-   c) nptII gene (complementary-to 185-988)-   d) 3′rbcL (complementary to 6-143)

The resulting vector was named pCB304-25 and also conferred kanamycinresistance to E. coli cells. This vector is no longer linearized bycommercially available I-PpoI. All of the insert of the vector pCB304-25(backbone pBluescript; replacing the MCS accordingly from SacI to KpnI)is described by SEQ ID NO: 62 and thus comprises the following elements:

-   a) Position bp 19 to 110: 3′psbA from tobacco-   b) Position bp 149 to 160: nonfunctional “half” of the I-PpoI    recognition sequence-   c) Position bp 171 to 277: Prps16 promoter-   d) Position bp 286 to 303: 5′rbcL sequence followed by 18 bp    encoding the first 6 amino acids of the rbcL protein (bp 304-321)-   e) Position bp 322 to 1125: nptII-   f) Position bp 1167 to 1304: 3′rbcL-   g) Position bp 1310 to 1319: nonfunctional “half” of the I-PpoI    recognition sequence    II) With I-PpoI in the Insertion Sequence

A BglII/MunI fragment which encoded a 5′psbA -1-PpoI fusion wasadditionally introduced into the vector pCB304-25 with the aid of theBamHI and EcoRI cleavage sites. The resulting vector pCB320-192 thusexpressed the nptII gene and I-PpoI homing endonuclease under thecontrol of the Prpsl6 promoter. The Bgl II/Mun I fragment is representedby SEQ ID NO: 63 and comprises the following elements:

-   a) Position bp 6 to 82: 5′psbA-   b) Position bp 83 to 574: I-PpoI    5.3 Generation of a Transformation Vector for Artificial Homing with    a Homologous Region Flanking the Insertion Sequence Unilaterally

The elements located upstream of the Prpsl6 promoter and which arehomologous to those in the master plants CB199NTH were removed from thevector pCB320-192 by restriction with KpnI and BglII. Instead, a BstXIcleavage site was introduced therein by means of syntheticoligonucleotides pl99 and p200.

p199 5′-GATCTCCAGTTAACTGGGGTAC-3′ (SEQ ID NO: 22) p2005′-CCCAGTTAACTGGA-3′ (SEQ ID NO: 23)

DNA ends which are compatible with those originating by restriction withI-PpoI can now be generated by cleaving with BstXI. The resulting vectorwas renamed pCB322-1. A fragment which, at its one side, has an endwhich is compatible with DNA which had been cleaved with I-PpoI at itscore recognition region and, at its other side, homology with plastomesequences of the master plants CB199NTH can be obtained from this vectorfor example using the enzymes BstXI and SacI.

5.4 Generation of a Transformation Vector for Artificial Homing WithoutHomologous Regions Around the Insertion Sequence

The remaining portion, which is homologous with recombinant plastidsequences of the master plants CB199NTH, was removed from the vectorpCB322-1 using SacI and BspTI. Simultaneously, a BstXI cleavage sitewhich, after cleavage with BstXI generates DNA ends which are compatiblewith I-PpoI-cut DNA, was generated here by introducing syntheticoligonucleotides p218 and p219. The resulting vector was namedpCB347-33.

(SEQ ID NO: 24) p218 5′-TTAAGCCAGTTAACTGGGCGGAGCT-3′ (SEQ ID NO: 25)p219 5′-CCGCCCAGTTAACTGGC-3′

A fragment with bilateral DNA ends which are compatible with the DNAends generated by the I-PpoI enzyme at its core recognition region canbe isolated from this vector using the enzyme BstXI.

Example 6 Use of the Master Plants CB199NTH for Plastid Transformationby Means of DSB Induction

6.1 Using the Vectors Generated in 5.1 and 5.2 for Transforming theMaster Plants CB199NTH by using the DSBI enzyme I-PpoI

The plasmid pCB304-24 and the plasmid pCB289-13 were simultaneouslyapplied to gold particles as described in Example 4 and used to bombardexplants of the master plants CB199NTH, which explants had been treatedanalogously to what has been said in Example 4. However, the procedurediffered from the decription in Example 4 insofar as incubation is firstcarried out for 10 days on the regeneration medium without antibiotics;later, kanamycin is used in a concentration of 50 mg/l (in contrast tothe 500 mg/l spectinomycin stated in Example 4).

The plasmid pCB320-192 was applied to gold particles as described inExample 4. After the ethanol washing step, 20 U of commerciallyavailable I-PpoI enzyme (Promega) were additionally added. Furthertreatment was as described above.

Also, in a different batch, 0.5 μg of a transcript generated in vitrowith the aid of the T7 polymerase was applied simultaneously with theplasmid pCB320-192 to the gold particles.

The template for the in-vitro transcription was HindIII-linearized DNAof the plasmid pCB289-13. The transcript generated thus thereforeencodes I-PpoI. After the bombardment, the treatment of the explants ofthe master plants continues as described above.

6.2 Using the Vectors Generated in 5.1 and 5.3 for Transforming theMaster Plants CB199NTH by Utilizing the DSBI Enzyme I-PpoI andHomologous Regions which are only Unilaterally Present

A fragment excised from the plasmid pCB322-1 with BstXI and SacI waseluted from an agarose gel. This fragment was subsequently applied togold particles simultaneously with 1 μg of in-vitro transcript ofHindIII-linearized plasmid pCB289-13 (cf. Example 6.1). After theexplants of the master plants CB199NTH have been bombarded, the rest ofthe treatment is as described for Example 6.1.

6.3 Using the Vectors Generated in 5.1 and 5.4 for Transforming theMaster Plants CB199NTH by Utilizing the DSBI Enzyme I-PpoI WithoutHomologous Regions

The insertion sequence was excised from the plasmid pCB347-33 by meansof BstXI and eluted from an agarose gel. This fragment was applied togold particles simultaneously with 1 μg of in-vitro transcript of theHindIII-linearized plasmid pCB289-13. The bombardment and the rest ofthe treatment are as detailed in Example 6.1.

Example 7 Identification of Naturally Occurring, Endogenous RecognitionRegions for Homing Endonucleases in Plastomes of Different Plant Species

Although no homing enconucleases are known to occur in the plastids ofhigher plants, known plastome sequences were tested for the presence ofrecognition regions for homing endonucleases. This was done with the aidof the computer program SeqMan II (DNASTAR Inc.). The recognitionregions which were identified in this manner are compiled in Table 1.

Based on the computer analysis, it was not possible to tell whetherI-SceI has a recognition region in the plastidic genome or not. Theregion which is most likely to be able to act as recognition region wasgenerated synthetically and integrated into the XbaI and XhoI cleavagesite of pBluescript in the form of oligonucleotides p276 and p277. Theresulting plasmid pCB414-1 was subsequently analyzed with the aid of acommercially available enzyme I-SceI (Roche) for the presence of afunctional cleavage site. The plasmid was indeed linearized by I-SceI.This leads to the conclusion that I-SceI which is expressed in lastidslikewise recognizes, and cleaves, this sequence. A further endogenousDSB recognition sequence for a DSBI enzyme has thus been identified.

(SEQ ID NO: 26) p276 5′-TCGAGAAGATCAGCCTGTTATCCCTAGAGTAACT-3′ (SEQ IDNO: 27) p277 5′-CTAGAGTTACTCTAGGGATAACAGGCTGATCTTC-3′

Example 8 Cloning Homologous Regions from the Tobacco Plastome whichFlank the Endogenous Recognition Region for the Homing EndonucleaseI-CpaI

DNA fragments from the 23S rDNA of the tobacco plastome were amplifiedby means of PCR upstream and downstream of the I-CpaI recognition regionusing the primers p93 and p97, and p98 and p95, respectively.

(SEQ ID NO: 64) p93: AAAGATCTCCTCACAAAGGGGGTCG (SEQ ID NO: 65) p97:TCGAAGACTTAGGACCGTTATAG (SEQ ID NO: 66) p98: AGGAAGACCTTGTCGGGTAAGTTCCG(SEQ ID NO: 67) p95: CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG

The resulting fragments were used for constructing the vector pCB270-1.The fragment from BssHII to BssHII of the pBlueScript vector (SEQ ID NO:8) is shown, the 5′ end of the sequence indicated being located at theBssHII cleavage site which is closer to the 3′ end of the lacZ gene.

Two BpiI cleavage sites were introduced between the DNA fragmentslocated upstream and downstream of the I-CpaI recognition regions. BpiIgenerates overhangs which are outside their recognition region. Thisprocedure ensured that the vector pCB270-1 could likewise be used forthe subsequent ingegration of various introns. To this end, simpleoverhangs which are compatible with the ends generated by BpiI in thevector pCB270-1 are generated at the introns to be cloned. Moreover, therespective nucleotides which are absent between the two fragments of the23S rDNA in the vector pCB270-1 are added onto the introns. The selectedregions are so highly conserved that there is no need to amplify newregions from other plant species. Furthermore, a point mutation in the23S rDNA, as has also been found in lincomycin-resistant mutants, hasbeen introduced into the sequence downstream of the I-CpaI recognitionregion via PCR strategy. The sequence of the vector pCB270-1 which hasbeen inserted into the pBluescript vector is shown in SEQ ID NO: 8. Thesequence comprises the following elements:

-   -   Fragment of the 23S rDNA upstream of the I-CpaI recognition        region: nucleotides 37 to 194    -   Fragment of the 23SrDNA upstream of the I-CpaI recognition        region: nucleotides 237 to 359    -   Point mutation for lincomycin resistance: 352 (A being native at        this point)

Vector pCB234-1 is constructed just as vector pCB270-1, but additionallyhas a recognition region for each of the restriction enconucleases XhoIand SacI downstream of the sequence shown hereinbelow.

Example 9 Cloning of the CpLSU2 Intron Including the Homing EndonucleaseI-CpaI

The CpLSU2 intron (SEQ ID NO: 15) was amplified from the DNA of the algaChlamydomonas pallidostigmatica (Culture Collection of Algae at theUniversity of Gbttingen, SAG Number 9.83, Chlamydomonas segnis,authentic strain of Chlamydomonas pallidostigmatica King) by means ofPCR using the oligonucleotides p91 and p92.

p91 5′-AGAAGACGATCCTAAGG-3′ (SEQ ID NO: 28) p925′-TGAAGACTTGACAAGGAATTTCGC-3′ (SEQ ID NO: 29)

The oligonucleotides were chosen in such a way that cloning into theBpiI cleavage sites of the vector pCB234-1 was possible, as describedabove. The sequence comprises the following elements:

-   -   Position 9-17—portion of the tobacco 23S rDNA which is absent in        pCB234-1    -   CpLSU2 intron: position 18-893    -   I-CpaI ORF: position 377-835    -   Position 894-909—portion of the tobacco 23S rDNA which is absent        in pCB234-1

This fragment, which comprises the CpLSU2 intron, was cloned into thebackbone of the vector pGEMTeasy (Promega) (vector pCB141-3). The entirefragment was excised from this vector using BpiI and cloned into theBpiI-linearized vector pCB234-1. The resulting vector was namedpCB254-2.

Example 10 Nuclear LSU-rRNA Intron from Tetrahymena thermophila

10.1 Cloning the LSU-rRNA Intron from Tetrahymena thermophila

The LSU-rRNA intron was amplified from the organism Tetrahymenathermophila by means of PCR. Again, the oligonucleotides p102 and p103were chosen in such a way that the nucleotides of the tobacco 23S rDNA,which are absent in pCB234-1, were added onto the intron to beamplified.

p102 (SEQ ID NO: 30): 5′-AGAAGACGATCCTAAATAGCAATATTTACCTTTGGGACCAAAAGTTATCAGGCATG-3′ p103 (SEQ ID NO: 31):5′TGAAGACTTGACAAGGAATTTCGCTACCTTCGAGTACTCCAAAACTAA TC-3′

Moreover, the internal guide sequence (underlined in p102) is mutated insuch a way over the wild type, owing to the choice of theoligonucleotide p102, that splicing of this intron-at the desiredposition in the tobacco 23S rDNA is possible. The sequence shown in SEQID NO: 7—the PCR fragment from the BpiI to BpiI cleavage site isshown—was cloned into the backbone of the vector pGEMTeasy. Theresulting vector was named pCB220-17. The sequence comprises thefollowing elements:

-   -   Position 9-12—portion of the tobacco 23S rDNA which is absent in        pCB234-1    -   LSU intron: position 13-425    -   Position 426-446—portion of the tobacco 23S rDNA which is absent        in pCB234-1

The Tetrahymena LSU intron including the added, flanking sequences, wasexcised from the vector pCB220-17 using BpiI and inserted into the BpiIcleavage sites of the vector pCB234-1. The resulting product was namedpCB255-1.

10.2 Indirect Detection of the Splicing Activity of the Tetrahymena LSUIntron in E. coli

To prove indirectly that the modified intron is indeed capable ofsplicing in the predetermined environment within the I-CpaI cleavagesite, the modified Tetrahymena intron from pCB220-17 together with aportion which surrounds the I-CpaI recognition region from the tobacco23S rDNA was cloned in such a way into the lacZ gene of pBluescriptthat, if this intron is spliced into E. coli (strain XL1-Blue), afunctional lacZ peptide can be formed. The expression of the latter canbe detected in suitable strains by methods with which the skilled workeris familiar by converting the substance5-bromo-4-chloro-3-indolyl-β_(D)-galactopyranoside (X-Gal) in the mediuminto a blue pigment. This vector was named pCB315-1. The lacZ geneincluding the introns in the vector pCB315-1 is described by SEQ ID NO:9. The vector backbone is identical with pBluescript. The sequencecomprises the following elements:

-   -   lacZ-5′ portion: complementary (789-765)    -   multiple cloning site from pBluescript: complementary (764-692)    -   23S rDNA fragment upstream and including the I-CpaI recognition        region: complementary (691-682)    -   modified Tetrahymena intron: complementary (681-269)    -   23S rDNA fragment upstream and including the I-CpaI recognition        region: complementary (268-244)    -   multiple cloning site from pBluescript: complementary (243-168)    -   lacZ-5′ portion: complementary (167-1)

A plasmid which corresponds to pCB315-1, but which plasmid (PCB305-1)lacks the element for the modified Tetrahymena intron, was generated forcontrol purposes. pCB305-1 thus acted as positive control to demonstratethat lacZ, with the tobacco plastome 23S rDNA nucleotides incorporatedin the reading frame is still functional. This reflects the situationafter correct splicing of the Tetrahymena intron. XL1-Blue competentcells were transformed with the plasmids pCB315-1 and pCB305-1 by meansof a method with which the skilled worker is familiar. In each case oneindividual colony was incubated on LB (Bactotryptone: 10 g/l, yeastextract: 5 g/l, NaCl: 10 g/l, pH 7.5) plates comprising 15 g/l Bactoagar, 40 μg/l ampicillin, 75 μg/l IPTG(isopropyl-β_(D)-thiogalactopyranoside) and 80 μg/l X-Gal overnight at37° C. In fact, both clones turned blue, which suggests that themodified Tetrahymena intron was spliced in the non-natural environmentof the tobacco 23S rDNA in the heterologous organismus E. coli.

10.3: Introduction of Further Sequences into the Tetrahymena LSU Intron

In addition to the experiments in Example 10.2, the possibility ofincorporating further elements into the modified Tetrahymena intronwithout destroying the splicing activity was studied. To this end,pCB315-1 was linearized with BglII and the overhangs were filled up withthe aid of Klenow fragment. Then, an XhoI-SacI fragment as is found inpCB199-3 was therefore cloned into this vector, likewise after treatmentwith Klenow fragment. A 229 bp fragment was thus inserted into themodified intron by this cloning step. This fragment comprises an I-PpoIrecognition region. Independently of the orientation in which the 229 bpfragment inserted into the Tetrahymena intron, a blue coloration wasdetected in the test as described in Example 10.2. This suggests thatthe Tetrahymena intron is capable both of splicing at the desired regionin the 23S rDNA and of incorporating additional genetic informationwhile nevertheless retaining a splicing activity.

10.4 Transformation of a Natural Master Plant and Destruction of theEndogenous I-CpaI Recognition Region with the Modified TetrahymenaIntron

The modified Tetrahymena intron was excised from the vector pCB220-17using BpiI and cloned into the BpiI linearized vector pCB234-1 asdescribed in principle in Example 8. The resulting vector was namedpCB255-1.

pCB255-1 is applied to gold particles simultaneously with in vitrotranscript of pCB262-5 (linearized with SalI, using T7 polymerase) bythe method described in Example 4. These gold particles are subsequentlyused to bombard tobacco plants cv. Petit Havana analogously to themethod described in Example 4. If appropriate, the explants can beselected on lincomycin (250 to 500 mg/l).

Example 11 Ll.LtrB Intron from Lactococcus lactis

The Ll.LtrB intron including few bases of the flanking exon sequenceswas amplified from Lactococcus lactis by means of PCR using the primersp207 and p208. The PCR product was cloned into the vector pCR2.1TA(Invitrogen) (pCB345-34) and sequenced (SEQ ID NO: 10). Few deviationsin comparison with the published sequence were found.

(SEQ ID NO: 32) p207 5′-GAGAAGACATTCCTAACACATCCATAACGTGCG-3′ (SEQ ID NO:33) p208 5′-TGAAGACTTGACATTTGATATGGTGAAGTAGG-3′

The cloned fragment in pCB345-34 (from the EcoRI cleavage site to theEcoRI cleavage site of the pCR2.1TA vector) is represented in SEQ ID NO:10. The remainder of the vector is identical with the backbone ofpCR2.1TA. The sequence comprises the following elements:

-   -   Portion of the natural 5′ exon: complementary (2540-2527)    -   Intron Ll.LtrB: complementary (2526-35)    -   ORF in the intron: complementary (1953-154)    -   Portion of the natural 3′ exon: complementary (34-28)

Example 12 Generation of a Further Derivative of the Tetrahymena LSUIntron, and Incorporation of a Foreign Gene into this Intron Derivative,and Transformation into Natural Master Plants

In accordance with a preferred embodiment of the present invention, anartificial intron was generated, which intron can be incorporated intothe plastidic genome at precisely the position where the natural intronbelonging to the DSB recognition sequence under investigation resides.In the present example, the Tetrahymena LSU intron was modified in sucha way that it is capable of splicing at the position marked “^” at therecognition site, identified within the scope of the present invention,for the DSBI enzyme I-CpaI in the plastidic genome of higher plants:CGATCCTAAGGT^AGCGAAATTCA.

The gene encoding I-CpaI including an RBS was subsequently incorporatedinto the intron. This gave rise to an intron which has splicingactivity, bears a foreign gene and which can be incorporated, by meansof the process found within the scope of the present invention, into theplastids of a natural master plant within an essential gene (encodingthe 23S rRNA).

12.1: Generation of a Further Tetrahymena LSU Intron Derivative

To obtain a functional intron derivative at a predefined insertion site,the internal guide sequence (IGS) must be adapted in such a way that itis capable of undergoing base pairing with the 5′ and the 3′ exon. FIG.10 illustrates how this adaptation was carried out in the presentexample in order to generate a Tetrahymena intron derivative which iscapable of splicing at the natural insertion site of the CpLSU5 intronwithin the I-CpaI recognition region. An adaptation to any desiredinsertion site can be carried out analogously. The intron generatedwithin the scope of the present example was named TetIVS2a and isdescribed by SEQ ID NO: 73.

12.2: Indirect Detection of the Splicing Activity of the TetIVS2a Intronin E. coli

TetIVS2a was incorporated into the lacz gene of pBluescript analogouslyto Example 10.2. After suitable incubation of E. coli XL1-blue cellswhich comprised the plasmid pCB459-1, a blue coloration indicated thesplicing activity of the TetIVS2a intron at the desired position.

Components of the insert from plasmid pCB459-1 (SEQ ID NO: 74; backbonecorresponds to pBluescript)

-   -   lacZ-3′ portion including parts of the multiple cloning site        from pBluescript (complementary to position 1-254)    -   Sequence from the I-CpaI recognition region (complementary to        position 254-265)    -   TetIVS2a (complementary to position 266-678)    -   Sequence from the I-CpaI recognition region (complementary to        position 679-687)    -   lacZ-5′ portion including parts of the multiple cloning site        from pBluescript (complementary to position 688-791)        12.3: Introduction of further Genetic Information into the        TetIVS2a Intron and Detection of the Splicing Activity in E.        coli

In this example, the gene encoding the DSBI enzyme I-CpaI is introducedinto TetIVS2a without the latter losing its splicing activity at saidposition within the I-CpaI recognition region.

To this end, a BclI cleavage site was first introduced, by PCR, into thesequence segment of TetIVS2a which corresponds to loop L8 in theTetrahymena LSU intron. A nonfunctional derivative of the gene encodingI-CpaI was then incorporated into this BclI cleavage site. Since theexpression of 1-CpaI in E. coli is toxic, it was necessary to use, forthe splice test in E. coli, a nonfunctional gene which had previouslybeen generated by linearizing the gene in question at the EcoRI cleavagesite, making the overhangs blunt-ended by Klenow treatment andsubsequently religating the gene segments. This resulted in areading-frame shift in the gene. Incorporation of said intron with thenonfunctional gene into the lacZ gene of pBluescript gave rise to theplasmid pCB478-3 and, again, it was possible to detect the splicingactivity of this intron in E. coli at the desired position within theI-CpaI recognition site by means of the blue coloration of colonies inquestion, analogously to Example 12.2. Since the functional geneencoding I-CpaI differs from the nonfunctional gene used in pCB478-3 byonly 4 bases, it can be assumed that the intron retains the desiredsplicing activity, even after the functional, instead of thenonfunctional, I-CpaI gene has been incorporated.

Components of the insert of plasmid pCB478-3 (SEQ ID NO: 75; backbonecorresponds to pBluescript)

-   -   lacZ-3′ portion including parts of the multiple cloning site        from pBluescript (complementary to position 1-265)    -   Sequence from the I-CpaI recognition region (complementary to        position 256-265)    -   TetIVS2a (complementary to position 266-1178), comprising a        nonfunctional gene for I-CpaI (complementary to position        399-861) and an RSB upstream of the nonfunctional I-CpaI gene        (complementary to 866-870)    -   Sequence from the I-CpaI recognition region (complementary to        position 1179-1187)    -   lacZ-5′ portion including parts of the multiple cloning site        from pBluescript (complementary to position 1179-1291)        12.4: Transformation of a Self-disseminating, Artificial Intron        in a Natural Master Plant

After it had been demonstrated that the TetIVS2a intron is capable ofsplicing at the desired position and of simultaneously incorporatingfurther genetic information without losing this splicing activity, aconstruct was generated which is intended to make possible that theI-CpaI gene can be introduced into the plastidic genome by means of themethod described within the scope of the present invention in the formof a foreign gene without using a selection marker. To this end, thevector pCB492-25, which comprises an insert with the following elementswas first generated (SEQ ID NO: 76; backbone corresponds to that ofpBluescript; sequence from BssHII to BssHII in pBluescript is indicated,the BssHII cleavage site indicated here at the 5′ end is the BssHIIcleavage site in pBluescript which is localized closer to the 3′ end ofthe lacZ gene):

-   -   23S rDNA fragment upstream of and including the I-CpaI        recognition region (position 37-203)    -   TetIVS2a (position 204-1112) with inserted gene encoding I-CpaI        (position 521-979) and RBS (position 512-516)    -   23S rDNA fragment downstream of and including the I-CpaI        recognition region (position 1113-1247)

To ensure expression of 1-CpaI directly after the introduction intoplastids of natural master plants, a promoter was added in vitroupstream of said intron Cpa construct by means of PCR. The primers p396and p95 and, as template, pCB492-25 were used for this purpose.

p396 (SEQ ID NO: 77): 5′-TAGTAAATGACAATTTTCCTCTGAATTATATAATTAACATGGCGACTGTTTACCAAAAAC-3 p95 (SEQ ID NO: 78):5′-CTCAATTGGGGTCTCTCTGTCCAGGTGCAGG-3′

The resulting PCR product was named Prom-TetIVS2a-Cpa, is described bySEQ ID NO: 79 and comprised the following elements:

-   -   synthetic promoter (position 8-40)    -   tobacco 23S rDNA upstream of and including portions of the        I-CpaI recognition region (position 41-207)    -   TetIVS2a (position 208-1116) comprising gene encoding I-CpaI        (position 525-983) and RBS (position 516-520)    -   tobacco 23S rDNA downstream of and including portions of the        I-CpaI recognition region (position 1117-1243)

The plasmid pCB492-25 was applied to gold particles simultaneously withthe above-described PCR product Prom-TetIVS2a-Cpa as described inExample 4 and subsequently used to bombard wild-type tobacco. Byexpression of the I-CpaI enzyme, it was intended to bring about adouble-strand break in the 23S rDNA, which double-strand break isrepaired by the PCR product which has been introduced or by theinsertion sequence, of the plasmid pCB492-25, which has been introduced.The I-CpaI recognition region, which is naturally present, is therebyinactivated in the transformed plastome copies. Plants were regeneratedwithout any selection pressure, and these plants are tested by PCR forthe presence of the insertion sequence in the plastome.

Example 13 Dissemination of the Modified Tetrahymena LSU Intron frompCB255-1 in a Natural Master Plant by Expression of the DSBI EnzymeI-CpaI in trans

This example shows how a DSBI enzyme can be expressed in the plastids ofmaster plants in order to efficiently disseminate an insertion sequencein the copies of the master plant.

13.1: Generation of a Vector for the Transformation of Plastids whichPermits the Expression of the Homing Endonuclease I-CpaI in Plastids

First, a vector which encodes the selection marker aadA and the DSBIenzyme I-CpaI was generated. Since expression of the I-CpaI enzyme islethal in E. coli, the accD promoter was chosen in order to allow forthe expression of this enzyme in the plastids, but to prevent theexpression in E. coli. Thus, it was possible to generate and amplifythis vector in a conventional manner with E. coli as the host organism.The resulting vector was named pCB435-45 and comprised an insert asshown in SEQ ID NO: 80 with the following elements:

-   -   Right-hand target region (as in pCB42-94, see above;        complementary to position 66-1403)    -   promoter PaccD (position 1422-1478)    -   RBS (position 1500-1504)    -   Gene encoding I-CpaI (position 1509-1967)    -   Expression cassette for the marker gene aadA consisting of:    -   the 3′ region of the psbA gene (complementary to position        2065-1974)    -   aadA gene (complementary to position 2872-2078)    -   5′-untranslated regions of the tobacco rbcL gene (complementary        to position 2890-2873), partly mutated    -   Promoter of the gene for the 16S rRNA (complementary to position        2987 to 2897)    -   Left-hand target region (as in pCB42-94, see above;        complementary to position 3863-3007)    -   Portions of the pBluescript (including origin of replication;        positions 3864-4746 and 1-65)        13.2 Cotransformation of pCB435-45 and pCB255-1 into Natural        Master Plants

The plasmids pCB435-45 and pCB255-1 were applied simultaneously to goldparticles as detailed in Example 4 and then introduced into plastids oftobacco leaves by means of the particle gun. Transplastomic plants wereselected on regeneration medium supplemented with 500 mg/l spectinomycinas described in Example 4. As soon as plantlets had formed, they weretransferred to growth medium supplemented with 500 mg/l spectinomycin,and leaf material was harvested. This leaf material was analyzed bySouthern analysis using the Dig-Easy Hyb® (Roche Diagnostics; Mannheim)for the incorporation of the two plasmids into the plastidic genome. Aprobe with a sequence as shown in SEQ ID NO: 81 was used to determinethe percentage of plastome copies which were transgenic with regard tothe insertion sequence from pCB435-45 (probe directed against portionsof the 16S rDNA).

A probe with a sequence as shown in SEQ ID NO: 82 was used to determinethe percentage of plastome copies which were transgenic with regard tothe insertion sequence from pCB255-1 (probe directed against portions ofthe 23S rDNA).

FIG. 11 shows that, in this experiment, 2 lines (CB255+435NTH-19 and-20) were identified which are transgenic with regard to both theinsertion sequence of pCB435-45 and that from pCB255-1. It wasfurthermore demonstrated in this manner that, surprisingly, theinsertion sequence from pCB255-1 (modified Tetrahymena LSU intron) hadalready been disseminated into more copies of the plastidic genome thanthe insertion sequence from pCB435-45, even though the selection hadbeen carried for the event of the insertion of the insertion sequencefrom pCB435-45 (aadA marker gene resides in pCB435-45). The efficiencyof the method described within the present invention—viz. the insertionand rapid dissemination of an insertion sequence in the plastidic genomewithout selecting for the presence of this insertion sequence byutilizing DSBI enzymes and suitable recognition sites—has thus beendemonstrated for said lines in the present example.

Example 14 Generation of Further Master Plants with a DSB RecognitionRegion which does not Naturally Occur in Plastids, and Transformation ofthese Plants Utilizing the DSBI Enzyme I-PpoI

14.1: Generation of a Further Vector (pCB456-2) for Introducing aNon-naturally-occurring Recognition Region for the Homing EndonucleaseI-PpoI into the Plastome of Tobacco

The purpose of this approach was (analogously to Example 3) to generatea further vector for the transformation of plastids, which vector has noextensive homologies with sequences in the plastidic genome.

In this plasmid, the selection marker aadA is under the control of asynthetic promoter which is derived from the consensus sequence for E.coli σ70 promoters. A region downstream of the Synechocystis 3′psbA-1gene was used as the 3′ end. In contrast to the vector pCB199-3 whichhas already been described, the DSB recognition sequence was hereintroduced into the molecule immediately downstream of the aadA gene,but upstream of the Synechocystis 3′psbA-1 sequence. An operon can begenerated thereby with the aid of a DSBI enzyme following insertion ofan insertion sequence. The genes on the insertion sequence can then beoptionally inserted on the insertion sequence without promoter. Afterthe insertion, suitable genes of the insertion sequence then also comeunder the control of the synthetic promoter upstream of the aadA gene inthe master plant. An operon structure consisting of the aadA and thesubsequently introduced genes can thereby optionally be generated in theplastome.

Various elements were cloned one after the other into the basic vectorpCB42-94 in order to generate the plasmid pCB456-2:

-   -   Synthetic promoter (complementary to bp 1226-1260)    -   Ribosome binding site (complementary to bp 1214-1218)    -   aadA gene (complementary to bp 414-1208)    -   Core recognition region for the homing endonuclease I-PpoI        (complementary to bp 331-345)    -   3′psbA-1 from Synechocystis (complementary to bp 19-155)

The vector thus obtained also confers spectinomycin resistance in E.coli. This vector, which is named pCB456-2, comprises the abovementionedelements within the nucleic acid sequence with the SEQ ID NO: 83,instead of the multiple cloning site in the basic vector pCB42-94 forthe transformation of plastids. Again, all of the sequence whichreplaces MCS (from SacI to KpnI) is indicated.

14.2: Generation of Predominantly Homotransplastomic Master Plants whichComprise a Nonnatural DSB Recognition Sequence

The vector pCB456-2 was introduced into the plastids of tobaccoanalogously to pCB199-3 in Example 4. However, as opposed to thedescription in Example 4, the shoots obtained were grown on growthmedium comprising 30 g/l sucrose (instead of the 10 g/l stated inExample 4). The resulting plants were named CB456NTH. 2 lines which havethe insertion sequence from pCB456-2 incorporated into their plastome(CB456NTH-1 and -15, cf. FIG. 12) were identified among thespectinomycin-resistant plants obtained after the transformation, usingSouthern hybridization. A probe which was directed against a fragment ofthe 16S rDNA was employed in the Southern experiment (cf. Example 13.2above). This probe was suitable for detecting an approx. 3.1 kb fragmentfrom EcoRI-digested DNA corresponding to the wild type. In contrast, anapprox. 1.7 kb fragment was detected when the insertion sequence frompCB456-2 had been incorporated into the corresponding plastome copies.

14.3: Generation of a Transformation Vector for Artificial Homing in theMaster Plants CB456NTH

First, an operon structure consisting of the elements RBS—nptII(encoding an enzyme which confers kanamycin resistance)—RBS —I-PpoI(encoding a DSBI enzyme) was generated. This cassette was surroundedwith BstXI cleavage sites which, after exposure to the enzyme BstXI,generate DNA ends which are compatible with the DNA ends generated bythe enzyme I-PpoI. The resulting vector (backbone corresponds to that ofpBluescript) was named pCB528-2 and comprises an insert as shown in SEQID NO: 84 with the following elements:

-   -   RBS (position 28-32)    -   nptII (position 27-840)    -   RBS (position 849-853)    -   Gene encoding I-PpoI (position 859-1350)

The 1360 bp fragment was subsequently excised from pCB528-2 using BstXIand ligated into the I-PpoI cleavage site in the vector pCB456-2. Cloneswith kanamycin resistance were selected from those obtained after theligation products had been transformed into E. coli. It was therebyensured that said insert in the clone in question was inserted in thevector in such a way that the nptII and I-PpoI cassettes had the sameorientation as the aadA cassette. This was also verified by therestriction analysis method, with which the skilled worker is familiar.The vector in question was named pCB535-11.

14.4: Transformation of pCB535-11 into Master Plants CB456NTH pCB535-11was Applied to Gold Particles as Described for pCB456-2 in Example 14.2and Subsequently Introduced into Plastids of the Master Plant CB456NTH-1Using the Particle Gun.

Some of the explants were incubated on regeneration medium without anyselection pressure. Resulting plants were transferred to growth medium(again without selection pressure). Thereafter, the plants are analyzedby PCR for the presence of the RBS—nptII—RBS—I-PpoI cassette.

Other explants were incubated on regeneration medium supplemented with15 or 30 mg/l kanamycin. After 2 weeks, the plants are transferred tofresh regeneration medium and the kanamycin concentration increasedstepwise to 50 and 80 mg/l, respectively.

1. A method for integration of a DNA sequence into a plastid DNA of aplant or plant cell, said method comprising: a) combining atransformation construct comprising an insertion sequence with at leastone enzyme suitable for directed induction of DNA double-strand breaksat a recognition sequence of the plastid DNA in at least one plastid ofa plant or plant cell, wherein the plastid DNA comprises at least onerecognition sequence for the directed induction of DNA double-strandbreaks; b) inducing DNA double-strand breaks at the recognitionsequence; and c) inserting the insertion sequence into the recognitionsequence of the plastid DNA at the DNA double-strand breaks, whereinfunctionality of the recognition sequence for the directed induction isdeactivated and said recognition sequence is no longer capable of beingcleaved by the at least one enzyme; wherein the at least one enzyme isexpressed in the plastid of a plant or plant cell or expressed in thenucleus and transported to the plastid of a plant or plant cell.
 2. Themethod of claim 1, wherein the recognition sequence and the insertionsequence are flanked at least unilaterally by sequences with sufficientlength and sufficient homology to ensure homologous recombination witheach other.
 3. The method of claim 1, wherein the transformationconstruct encompasses at least one element selected from the groupconsisting of: i) an expression cassette for an enzyme suitable for theinduction of DNA double-strand breaks at the recognition sequence forthe directed induction of DNA double-strand breaks; ii) a positiveselection marker; iii) a negative selection marker; iv) a reporter gene;v) a replication origin; vi) a multiple cloning region; vii) a sequencewhich makes possible homologous recombination or insertion into thegenome of a host organism; and viii) combinations thereof.
 4. The methodof claim 1, wherein the enzyme is selected from the group consisting ofrestriction endonucleases and homing endonucleases.
 5. The method ofclaim 1, wherein the at least one enzyme is selected from the group ofhoming endonucleases consisting of F-SceI, F-SceII, F-SuvI, F-TevI,F-TevII, I-AmaI, I-AniI, I-CeuI, I-CeuAIIP, I-ChuI, I-CmoeI, I-CpaI,I-CpaII, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP,I-CsmI, I-CvuI, I-CvuAIP, I-DdiII, I-DirI, I-DmoI, I-HspNIP, I-LlaI,I-MsoI, I-NaaI, I-NanI, I-NclIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp2361P,I-PakI, I-Pbo1P, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI,I-PorIIP, I-PpbIP, I-PpoI, I-SPBetaIP, I-ScaI, I-SceI, I-SceII,I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-SexIP, I-SneIP,I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP,I-SthPhiST3P, I-SthPhiS3bP, I-Tde1P, I-TevI, I-TevII, I-TevIII, I-UarAP,I-UarHGPA1P, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP,PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-PspI,PI-Rma43812IP, PI SPBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI,PI-TliI, and PI-TliII.
 6. The method of claim 1, wherein the at leastone enzyme is selected from the group consisting of enzymes that containthe amino acid sequence of SEQ ID NO: 5, 12, 14 or
 71. 7. The method ofclaim 1, wherein the at least one enzyme is expressed from an expressioncassette.
 8. The method of claim 1, wherein the plastid DNA is derivedfrom a cell of a multi-celled plant.
 9. The method of claim 1, furthercomprising isolating a plant or plant cell in which the insertionsequence has been inserted into the plastid DNA in at least one plastidof the plant or plant cell.
 10. The method of claim 9, furthercomprising selecting a predominantly homotranspiastomic plant cellcontaining the insertion sequence inserted into its plastid DNA.
 11. Amulti-celled plant formed from the predominantly homotransplastomicplant cell selected by the method of claim
 10. 12. The multi-celledplant of claim 11, further comprising an expression cassette insertedinto the plastid DNA.
 13. The multi-celled plant of claim 11, furthercomprising an expression cassette inserted into the nuclear DNA of saidplant cell.
 14. The multi-celled plant of claim 11, wherein the plant isselected from the group consisting of Arabidopsis thaliana, tobacco,Tagetes, wheat, rye, barley, oats, oilseed rape, maize, potato, sugarbeet, soybean, sunflower, pumpkin/squash and peanut.
 15. A cell culture,organ, tissue, part or transgenic propagation material derived from themulti-celled plant of claim 11, wherein the cell culture, organ, tissue,part or transgenic propagation material comprises the insertionsequence.
 16. A pharmaceutical, fine chemical, food, feed or seed,comprising the cell culture, organ, part or transgenic propagationmaterial of claim 15.